Modulation of prekallikrein (pkk) expression

ABSTRACT

Disclosed herein are antisense compounds and methods for decreasing PKK mRNA and protein expression. Such methods, compounds, and compositions are useful to treat, prevent, or ameliorate PKK-associated diseases, disorders, and conditions.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBIOL0172USC2SEQ_ST25.txt created Sep. 7, 2018, which is approximately632 KB in size. The information in the electronic format of the sequencelisting is incorporated herein by reference in its entirety.

FIELD

Provided are compounds, compositions, and methods for reducingexpression of human plasma prekallikrein (PKK) mRNA and protein in ananimal. Such compositions and methods are useful to treat, prevent, orameliorate inflammatory and thromboembolic conditions.

BACKGROUND

Plasma prekallikrein (PKK) is the precursor of plasma kallikrein (PK),which is encoded by the KLKB1 gene. PKK is a glycoprotein thatparticipates in the surface-dependent activation of blood coagulation,fibrinolysis, kinin generation, and inflammation. PKK is converted to PKby Factor XIIa by the cleavage of an internal Arg-Ile peptide bond. PKliberates kinins from kininogens and also generates plasmin fromplasminogen. PK is a member of the kinin-kallikrein pathway, whichconsists of several proteins that play a role in inflammation, bloodpressure control, coagulation, and pain.

SUMMARY

Provided herein are compounds, compositions, and methods for modulatingexpression of PKK mRNA and protein. In certain embodiments, compoundsuseful for modulating expression of PKK mRNA and protein are antisensecompounds. In certain embodiments, the antisense compounds are antisenseoligonucleotides.

In certain embodiments, modulation can occur in a cell or tissue. Incertain embodiments, the cell or tissue is in an animal. In certainembodiments, the animal is a human. In certain embodiments, PKK mRNAlevels are reduced. In certain embodiments, PKK protein levels arereduced. Such reduction can occur in a time-dependent manner or in adose-dependent manner.

Also provided are compounds, compositions, and methods useful forpreventing, treating, and ameliorating diseases, disorders, andconditions associated with PKK. In certain embodiments, such PKKassociated diseases, disorders, and conditions are inflammatorydiseases. In certain embodiments, the inflammatory disease may be anacute or chronic inflammatory disease. In certain embodiments, suchinflammatory diseases may include hereditary angioedema (HAE), edema,angioedema, swelling, angioedema of the lids, ocular edema, macularedema, and cerebral edema. In certain embodiments, such PKK associateddiseases, disorders, and conditions are thromboembolic diseases. Incertain embodiments, such thromboembolic diseases may includethrombosis, embolism, thromboembolism, deep vein thrombosis, pulmonaryembolism, myocardial infarction, stroke, and infarct.

Such diseases, disorders, and conditions can have one or more riskfactors, causes, or outcomes in common.

Certain risk factors and causes for development of an inflammatorydisease include genetic predisposition to an inflammatory disease andenvironmental factors. In certain embodiments, the subject has a mutatedcomplement 1 esterase inhibitor (C1-INH) gene or mutated Factor 12 gene.In certain embodiments, the subject has taken or is onangiotensin-converting enzyme inhibitors (ACE inhibitors) or angiotensinII receptor blockers (ARBs). In certain embodiments, the subject has hadan allergic reaction leading to angioedema. In certain embodiments, thesubject has type I HAE. In certain embodiments, the subject has type IIHAE. In certain embodiments, the subject has type III HAE.

Certain outcomes associated with development of an inflammatory diseaseinclude edema/swelling in various body parts including the extremities(i.e., hands, feet, arms, legs), the intestines (abdomen), the face, thegenitals, the larynx (i.e., voice box); vascular permeability; vascularleakage; generalized inflammation; abdominal pain; bloating; vomiting;diarrhea; itchy skin; respiratory (asthmatic) reactions; rhinitis;anaphylaxis; bronchoconstriction; hypotension; coma; and death.

Certain risk factors and causes for development of a thromboembolicdisease include genetic predisposition to a thromboembolic disease,immobility, surgery (particularly orthopedic surgery), malignancy,pregnancy, older age, use of oral contraceptives, atrial fibrillation,previous thromboembolic condition, chronic inflammatory disease, andinherited or acquired prothrombotic clotting disorders. Certain outcomesassociated with development of a thromboembolic condition includedecreased blood flow through an affected vessel, death of tissue, anddeath.

In certain embodiments, methods of treatment include administering a PKKantisense compound to an individual in need thereof. In certainembodiments, methods of treatment include administering a PKK antisenseoligonucleotide to an individual in need thereof.

LISTING OF FIGURES

FIG. 1 is a Western blot quantification of HMWK from blood samples asdescribed in Example 11.

FIG. 2 is a Western blot quantification of HMWK from blood samples asdescribed in Example 14.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Herein, the use ofthe singular includes the plural unless specifically stated otherwise.As used herein, the use of “or” means “and/or” unless stated otherwise.Furthermore, the use of the term “including” as well as other forms,such as “includes” and “included”, is not limiting. Also, terms such as“element” or “component” encompass both elements and componentscomprising one unit and elements and components that comprise more thanone subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference forthe portions of the document discussed herein, as well as in theirentirety.

Definitions

Unless specific definitions are provided, the nomenclature utilized inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, andchemical analysis. Where permitted, all patents, applications, publishedapplications and other publications, GENBANK Accession Numbers andassociated sequence information obtainable through databases such asNational Center for Biotechnology Information (NCBI) and other datareferred to throughout in the disclosure herein are incorporated byreference for the portions of the document discussed herein, as well asin their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

“2′-O-methoxyethyl” (also 2′-MOE and 2′-OCH₂CH₂—OCH₃ and MOE) refers toan O-methoxyethyl modification of the 2′ position of a furanose ring. A2′-O-methoxyethyl modified sugar is a modified sugar.

“2′-O-methoxyethyl modified nucleoside” (also “2′-MOE nucleoside”) meansa nucleoside comprising a 2′-MOE modified sugar moiety.

“2′-substituted nucleoside” means a nucleoside comprising a substituentat the 2′-position of the furanose ring other than H or OH. In certainembodiments, 2′ substituted nucleosides include nucleosides withbicyclic sugar modifications.

“2′-deoxynucleoside” means a nucleoside comprising a hydrogen at the 2′position of the sugar portion of the nucleoside.

“3′ target site” refers to the nucleotide of a target nucleic acid whichis complementary to the 3′-most nucleotide of a particular antisensecompound.

“5′ target site” refers to the nucleotide of a target nucleic acid whichis complementary to the 5′-most nucleotide of a particular antisensecompound.

“5-methylcytosine” means a cytosine modified with a methyl groupattached to the 5 position. A 5-methylcytosine is a modified nucleobase.

“About” means within +7% of a value. For example, if it is stated, “thecompounds affected at least about 70% inhibition of PKK”, it is impliedthat the PKK levels are inhibited within a range of 63% and 77%.

“Administered concomitantly” refers to the co-administration of twopharmaceutical agents in any manner in which the pharmacological effectsof both are manifest in the patient at the same time. Concomitantadministration does not require that both pharmaceutical agents beadministered in a single pharmaceutical composition, in the same dosageform, or by the same route of administration. The effects of bothpharmaceutical agents need not manifest themselves at the same time. Theeffects need only be overlapping for a period of time and need not becoextensive.

“Administering” means providing a pharmaceutical agent to an animal, andincludes, but is not limited to administering by a medical professionaland self-administering.

“Amelioration” refers to a lessening, slowing, stopping, or reversing ofat least one indicator of the severity of a condition or disease. Theseverity of indicators may be determined by subjective or objectivemeasures, which are known to those skilled in the art.

“Animal” refers to a human or non-human animal, including, but notlimited to, mice, rats, rabbits, dogs, cats, pigs, and non-humanprimates, including, but not limited to, monkeys and chimpanzees.

“Antisense activity” means any detectable or measurable activityattributable to the hybridization of an antisense compound to its targetnucleic acid. In certain embodiments, antisense activity is a decreasein the amount or expression of a target nucleic acid or protein encodedby such target nucleic acid. “Antisense compound” means an oligomericcompound that is capable of undergoing hybridization to a target nucleicacid through hydrogen bonding. Examples of antisense compounds includesingle-stranded and double-stranded compounds, such as, antisenseoligonucleotides, siRNAs, shRNAs, ssRNAs, and occupancy-based compounds.

“Antisense compound” means an oligomeric compound that is capable ofundergoing hybridization to a target nucleic acid through hydrogenbonding. Examples of antisense compounds include single-stranded anddouble-stranded compounds, such as, antisense oligonucleotides, siRNAs,shRNAs, ssRNAs, and occupancy-based compounds.

“Antisense inhibition” means reduction of target nucleic acid levels inthe presence of an antisense compound complementary to a target nucleicacid compared to target nucleic acid levels or in the absence of theantisense compound. “Antisense mechanisms” are all those mechanismsinvolving hybridization of a compound with target nucleic acid, whereinthe outcome or effect of the hybridization is either target degradationor target occupancy with concomitant stalling of the cellular machineryinvolving, for example, transcription or splicing.

“Antisense mechanisms” are all those mechanisms involving hybridizationof a compound with a target nucleic acid, wherein the outcome or effectof the hybridization is either target degradation or target occupancywith concomitant stalling of the cellular machinery involving, forexample, transcription or splicing.

“Antisense oligonucleotide” means a single-stranded oligonucleotidehaving a nucleobase sequence that permits hybridization to acorresponding segment of a target nucleic acid. “Base complementarity”refers to the capacity for the precise base pairing of nucleobases of anantisense oligonucleotide with corresponding nucleobases in a targetnucleic acid (i.e., hybridization), and is mediated by Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen binding between correspondingnucleobases.

“Base complementarity” refers to the capacity for the precise basepairing of nucleobases of an antisense oligonucleotide withcorresponding nucleobases in a target nucleic acid (i.e.,hybridization), and is mediated by Watson-Crick, Hoogsteen or reversedHoogsteen hydrogen binding between corresponding nucleobases.

“Bicyclic sugar” means a furanose ring modified by the bridging of twoatoms. A bicyclic sugar is a modified sugar.

“Bicyclic nucleoside” (also bicyclic nucleic acid or BNA) means anucleoside having a sugar moiety comprising a bridge connecting twocarbon atoms of the sugar ring, thereby forming a bicyclic ring system.In certain embodiments, the bridge connects the 4′-carbon and the2′-carbon of the sugar ring.

“Cap structure” or “terminal cap moiety” means chemical modifications,which have been incorporated at either terminus of an antisensecompound.

“cEt” or “constrained ethyl” means a bicyclic nucleoside having a sugarmoiety comprising a bridge connecting the 4′-carbon and the 2′-carbon,wherein the bridge has the formula: 4′-CH(CH₃)—O-2′.

“cEt modified nucleoside” (also “constrained ethyl nucleoside”) means anucleoside comprising a bicyclic sugar moiety comprising a4′-CH(CH₃)—O-2′ bridge.

“Chemically distinct region” refers to a region of an antisense compoundthat is in some way chemically different than another region of the sameantisense compound. For example, a region having 2′-O-methoxyethylnucleosides is chemically distinct from a region having nucleosideswithout 2′-O-methoxyethyl modifications.

“Chimeric antisense compound” means an antisense compound that has atleast two chemically distinct regions, each position having a pluralityof subunits.

“Co-administration” means administration of two or more pharmaceuticalagents to an individual. The two or more pharmaceutical agents may be ina single pharmaceutical composition, or may be in separatepharmaceutical compositions. Each of the two or more pharmaceuticalagents may be administered through the same or different routes ofadministration. Co-administration encompasses parallel or sequentialadministration.

“Complementarity” means the capacity for pairing between nucleobases ofa first nucleic acid and a second nucleic acid.

“Comprise,” “comprises,” and “comprising” will be understood to implythe inclusion of a stated step or element or group of steps or elementsbut not the exclusion of any other step or element or group of steps orelements.

“Contiguous nucleobases” means nucleobases immediately adjacent to eachother.

“Designing” or “Designed to” refer to the process of creating anoligomeric compound that specifically hybridizes with a selected nucleicacid molecule.

“Diluent” means an ingredient in a composition that lackspharmacological activity, but is pharmaceutically necessary ordesirable. For example, in drugs that are injected, the diluent may be aliquid, e.g. saline solution.

“Dose” means a specified quantity of a pharmaceutical agent provided ina single administration, or in a specified time period. In certainembodiments, a dose may be administered in one, two, or more boluses,tablets, or injections. For example, in certain embodiments wheresubcutaneous administration is desired, the desired dose requires avolume not easily accommodated by a single injection, therefore, two ormore injections may be used to achieve the desired dose. In certainembodiments, the pharmaceutical agent is administered by infusion overan extended period of time or continuously. Doses may be stated as theamount of pharmaceutical agent per hour, day, week, or month.

“Downstream” refers to the relative direction toward the 3′ end orC-terminal end of a nucleic acid.

“Effective amount” in the context of modulating an activity or oftreating or preventing a condition means the administration of thatamount of pharmaceutical agent to a subject in need of such modulation,treatment, or prophylaxis, either in a single dose or as part of aseries, that is effective for modulation of that effect, or fortreatment or prophylaxis or improvement of that condition. The effectiveamount may vary among individuals depending on the health and physicalcondition of the individual to be treated, the taxonomic group of theindividuals to be treated, the formulation of the composition,assessment of the individual's medical condition, and other relevantfactors.

“Efficacy” means the ability to produce a desired effect.

“Expression” includes all the functions by which a gene's codedinformation is converted into structures present and operating in acell. Such structures include, but are not limited to the products oftranscription and translation.

“Fully complementary” or “100% complementary” means each nucleobase of afirst nucleic acid has a complementary nucleobase in a second nucleicacid. In certain embodiments, a first nucleic acid is an antisensecompound and a target nucleic acid is a second nucleic acid.

“Gapmer” means a chimeric antisense compound in which an internal regionhaving a plurality of nucleosides that support RNase H cleavage ispositioned between external regions having one or more nucleosides,wherein the nucleosides comprising the internal region are chemicallydistinct from the nucleoside or nucleosides comprising the externalregions. The internal region may be referred to as a “gap” and theexternal regions may be referred to as the “wings.”

“Hybridization” means the annealing of complementary nucleic acidmolecules. In certain embodiments, complementary nucleic acid moleculesinclude, but are not limited to, an antisense compound and a targetnucleic acid. In certain embodiments, complementary nucleic acidmolecules include, but are not limited to, an antisense oligonucleotideand a nucleic acid target.

“Identifying an animal having an inflammatory disease” means identifyingan animal having been diagnosed with an inflammatory disease orpredisposed to develop an inflammatory disease. Individuals predisposedto develop an inflammatory disease include those having one or more riskfactors for developing an inflammatory disease including environmentalfactors, having a personal or family history, or genetic predispositionto one or more inflammatory disease. Such identification may beaccomplished by any method including evaluating an individual's medicalhistory and standard clinical tests or assessments, such as genetictesting.

“Identifying an animal having a PKK associated disease” meansidentifying an animal having been diagnosed with a PKK associateddisease or predisposed to develop a PKK associated disease. Individualspredisposed to develop a PKK associated disease include those having oneor more risk factors for developing a PKK associated disease includinghaving a personal or family history, or genetic predisposition of one ormore PKK associated diseases. Such identification may be accomplished byany method including evaluating an individual's medical history andstandard clinical tests or assessments, such as genetic testing.

“Identifying an animal having a thromboembolic disease” meansidentifying an animal having been diagnosed with a thromboembolicdisease or predisposed to develop a thromboembolic disease. Individualspredisposed to develop a thromboembolic disease include those having oneor more risk factors for developing a thromboembolic disease includinghaving a personal or family history, or genetic predisposition of one ormore thromboembolic diseases, immobility, surgery (particularlyorthopedic surgery), malignancy, pregnancy, older age, use of oralcontraceptives, atrial fibrillation, previous thromboembolic condition,chronic inflammatory disease, and inherited or acquired prothromboticclotting disorders. Such identification may be accomplished by anymethod including evaluating an individual's medical history and standardclinical tests or assessments, such as genetic testing.

“Immediately adjacent” means there are no intervening elements betweenthe immediately adjacent elements. “Individual” means a human ornon-human animal selected for treatment or therapy.

“Individual” means a human or non-human animal selected for treatment ortherapy.

“Inhibiting PKK” means reducing the level or expression of a PKK mRNAand/or protein. In certain embodiments, PKK mRNA and/or protein levelsare inhibited in the presence of an antisense compound targeting PKK,including an antisense oligonucleotide targeting PKK, as compared toexpression of PKK mRNA and/or protein levels in the absence of a PKKantisense compound, such as an antisense oligonucleotide.

“Inhibiting the expression or activity” refers to a reduction orblockade of the expression or activity and does not necessarily indicatea total elimination of expression or activity.

“Internucleoside linkage” refers to the chemical bond betweennucleosides.

“Linked nucleosides” means adjacent nucleosides linked together by aninternucleoside linkage.

“Locked nucleic acid” or “LNA” or “LNA nucleosides” means nucleic acidmonomers having a bridge connecting two carbon atoms between the 4′ and2′position of the nucleoside sugar unit, thereby forming a bicyclicsugar. Examples of such bicyclic sugar include, but are not limited toA) α-L-Methyleneoxy (4′-CH₂—O-2′) LNA, (B) β-D-Methyleneoxy(4′-CH₂—O-2′) LNA, (C) Ethyleneoxy (4′-(CH₂)₂—O-2′) LNA, (D) Aminooxy(4′-CH₂—O—N(R)-2′) LNA and (E) Oxyamino (4′-CH₂—N(R)—O-2′) LNA, asdepicted below.

As used herein, LNA compounds include, but are not limited to, compoundshaving at least one bridge between the 4′ and the 2′ position of thesugar wherein each of the bridges independently comprises 1 or from 2 to4 linked groups independently selected from —[C(R₁)(R₂)]_(n)—,—C(R₁)═C(R₂)—, —C(R₁)═N—, —C(═NR₁)—, —C(═O)—, —C(═S)—, —O—, —Si(R₁)₂—,—S(═O)— and —N(R₁)—; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; eachR₁ and R₂ is, independently, H, a protecting group, hydroxyl, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, a heterocycle radical, a substitutedheterocycle radical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclicradical, substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁,N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁),or sulfoxyl (S(═O)-J₁); and each J₁ and J₂ is, independently, H, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, acyl (C(═O)—H), substituted acyl, a heterocycleradical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl,substituted C₁-C₁₂ aminoalkyl or a protecting group.

Examples of 4′-2′ bridging groups encompassed within the definition ofLNA include, but are not limited to one of formulae: —[C(R₁)(R₂)]_(n)—,—[C(R₁)(R₂)]_(n)—O—, —C(R₁R₂)—N(R₁)—O— or —C(R₁R₂)—O—N(R₁)—.Furthermore, other bridging groups encompassed with the definition ofLNA are 4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′, 4′-CH₂—O-2′,4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R₁)-2′ and 4′-CH₂—N(R₁)—O-2′- bridges,wherein each R₁ and R₂ is, independently, H, a protecting group orC₁-C₁₂ alkyl.

Also included within the definition of LNA according to the inventionare LNAs in which the 2′-hydroxyl group of the ribosyl sugar ring isconnected to the 4′ carbon atom of the sugar ring, thereby forming amethyleneoxy (4′-CH₂—O-2′) bridge to form the bicyclic sugar moiety. Thebridge can also be a methylene (—CH₂—) group connecting the 2′ oxygenatom and the 4′ carbon atom, for which the term methyleneoxy(4′-CH₂—O-2′) LNA is used. Furthermore; in the case of the bicyclicsugar moiety having an ethylene bridging group in this position, theterm ethyleneoxy (4′-CH₂CH₂—O-2′) LNA is used. α-L-methyleneoxy(4′-CH₂—O-2′), an isomer of methyleneoxy (4′-CH₂—O-2′) LNA is alsoencompassed within the definition of LNA, as used herein.

“Mismatch” or “non-complementary nucleobase” refers to the case when anucleobase of a first nucleic acid is not capable of pairing with thecorresponding nucleobase of a second or target nucleic acid.

“Modified intemucleoside linkage” refers to a substitution or any changefrom a naturally occurring intemucleoside bond (i.e. a phosphodiesterintemucleoside bond).

“Modified nucleobase” means any nucleobase other than adenine, cytosine,guanine, thymidine (also known as 5-methyluracil), or uracil. An“unmodified nucleobase” means the purine bases adenine (A) and guanine(G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

“Modified nucleoside” means a nucleoside having, independently, amodified sugar moiety and/or modified nucleobase.

“Modified nucleotide” means a nucleotide having, independently, amodified sugar moiety, modified intemucleoside linkage, and/or modifiednucleobase.

“Modified oligonucleotide” means an oligonucleotide comprising at leastone modified intemucleoside linkage, modified sugar, and/or modifiednucleobase.

“Modified sugar” means substitution and/or any change from a naturalsugar moiety.

“Monomer” means a single unit of an oligomer. Monomers include, but arenot limited to, nucleosides and nucleotides, whether naturally occurringor modified.

“Motif” means the pattern of unmodified and modified nucleosides in anantisense compound.

“Natural sugar moiety” means a sugar moiety found in DNA (2′-H) or RNA(2′-OH).

“Naturally occurring internucleoside linkage” means a 3′ to 5′phosphodiester linkage.

“Non-complementary nucleobase” refers to a pair of nucleobases that donot form hydrogen bonds with one another or otherwise supporthybridization.

“Nucleic acid” refers to molecules composed of monomeric nucleotides. Anucleic acid includes, but is not limited to, ribonucleic acids (RNA),deoxyribonucleic acids (DNA), single-stranded nucleic acids,double-stranded nucleic acids, small interfering ribonucleic acids(siRNA), and microRNAs (miRNA).

“Nucleobase” means a heterocyclic moiety capable of pairing with a baseof another nucleic acid.

“Nucleobase complementarity” refers to a nucleobase that is capable ofbase pairing with another nucleobase. For example, in DNA, adenine (A)is complementary to thymine (T). For example, in RNA, adenine (A) iscomplementary to uracil (U). In certain embodiments, complementarynucleobase refers to a nucleobase of an antisense compound that iscapable of base pairing with a nucleobase of its target nucleic acid.For example, if a nucleobase at a certain position of an antisensecompound is capable of hydrogen bonding with a nucleobase at a certainposition of a target nucleic acid, then the position of hydrogen bondingbetween the oligonucleotide and the target nucleic acid is considered tobe complementary at that nucleobase pair.

“Nucleobase sequence” means the order of contiguous nucleobasesindependent of any sugar, linkage, and/or nucleobase modification.

“Nucleoside” means a nucleobase linked to a sugar.

“Nucleoside mimetic” includes those structures used to replace the sugaror the sugar and the base and not necessarily the linkage at one or morepositions of an oligomeric compound such as for example nucleosidemimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl,bicyclo, or tricyclo sugar mimetics, e.g., non furanose sugar units.Nucleotide mimetic includes those structures used to replace thenucleoside and the linkage at one or more positions of an oligomericcompound such as for example peptide nucleic acids or morpholinos(morpholinos linked by —N(H)—C(═O)—O— or other non-phosphodiesterlinkage). Sugar surrogate overlaps with the slightly broader termnucleoside mimetic but is intended to indicate replacement of the sugarunit (furanose ring) only. The tetrahydropyranyl rings provided hereinare illustrative of an example of a sugar surrogate wherein the furanosesugar group has been replaced with a tetrahydropyranyl ring system.“Mimetic” refers to groups that are substituted for a sugar, anucleobase, and/or intemucleoside linkage. Generally, a mimetic is usedin place of the sugar or sugar-internucleoside linkage combination, andthe nucleobase is maintained for hybridization to a selected target.

“Nucleotide” means a nucleoside having a phosphate group covalentlylinked to the sugar portion of the nucleoside.

“Off-target effect” refers to an unwanted or deleterious biologicaleffect associated with modulation of RNA or protein expression of a geneother than the intended target nucleic acid.

“Oligomeric compound” or “oligomer” means a polymer of linked monomericsubunits which is capable of hybridizing to at least a region of anucleic acid molecule.

“Oligonucleotide” means a polymer of linked nucleosides each of whichcan be modified or unmodified, independent one from another.

“Parenteral administration” means administration through injection(e.g., bolus injection) or infusion. Parenteral administration includessubcutaneous administration, intravenous administration, intramuscularadministration, intraarterial administration, intraperitonealadministration, or intracranial administration, e.g., intrathecal orintracerebroventricular administration.

“Peptide” means a molecule formed by linking at least two amino acids byamide bonds. Without limitation, as used herein, peptide refers topolypeptides and proteins.

“Pharmaceutical agent” means a substance that provides a therapeuticbenefit when administered to an individual. For example, in certainembodiments, an antisense oligonucleotide targeted to PKK is apharmaceutical agent.

“Pharmaceutical composition” means a mixture of substances suitable foradministering to a subject. For example, a pharmaceutical compositionmay comprise an antisense oligonucleotide and a sterile aqueoussolution.

“Pharmaceutically acceptable derivative” encompasses pharmaceuticallyacceptable salts, conjugates, prodrugs or isomers of the compoundsdescribed herein.

“Pharmaceutically acceptable salts” means physiologically andpharmaceutically acceptable salts of antisense compounds, i.e., saltsthat retain the desired biological activity of the parentoligonucleotide and do not impart undesired toxicological effectsthereto.

“Phosphorothioate linkage” means a linkage between nucleosides where thephosphodiester bond is modified by replacing one of the non-bridgingoxygen atoms with a sulfur atom. A phosphorothioate linkage is amodified intemucleoside linkage.

“PKK” means mammalian plasma prekallikrein, including human plasmaprekallikrein. Plasma prekallikrein (PKK) is the precursor of plasmakallikrein (PK), which is encoded by the KLKB1 gene.

“PKK associated disease” means any disease associated with any PKKnucleic acid or expression product thereof. Such diseases may include aninflammatory disease or a thromboembolic disease. Such diseases mayinclude hereditary angioedema (HAE).

“PKK mRNA” means any messenger RNA expression product of a DNA sequenceencoding PKK.

“PKK nucleic acid” means any nucleic acid encoding PKK. For example, incertain embodiments, a PKK nucleic acid includes a DNA sequence encodingPKK, an RNA sequence transcribed from DNA encoding PKK (includinggenomic DNA comprising introns and exons), and an mRNA sequence encodingPKK. “PKK mRNA” means an mRNA encoding a PKK protein.

“PKK protein” means the polypeptide expression product of a PKK nucleicacid.

“Portion” means a defined number of contiguous (i.e., linked)nucleobases of a nucleic acid. In certain embodiments, a portion is adefined number of contiguous nucleobases of a target nucleic acid. Incertain embodiments, a portion is a defined number of contiguousnucleobases of an antisense compound.

“Prevent” or “preventing” refers to delaying or forestalling the onsetor development of a disease, disorder, or condition for a period of timefrom minutes to days, weeks to months, or indefinitely.

“Prodrug” means a therapeutic agent that is prepared in an inactive formthat is converted to an active form (i.e., drug) within the body orcells thereof by the action of endogenous enzymes or other chemicalsand/or conditions.

“Prophylactically effective amount” refers to an amount of apharmaceutical agent that provides a prophylactic or preventativebenefit to an animal.

“Region” is defined as a portion of the target nucleic acid having atleast one identifiable structure, function, or characteristic.

“Ribonucleotide” means a nucleotide having a hydroxy at the 2′ positionof the sugar portion of the nucleotide. Ribonucleotides may be modifiedwith any of a variety of substituents.

“Salts” mean a physiologically and pharmaceutically acceptable salts ofantisense compounds, i.e., salts that retain the desired biologicalactivity of the parent oligonucleotide and do not impart undesiredtoxicological effects thereto.

“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid.

“Side effects” means physiological responses attributable to a treatmentother than desired effects. In certain embodiments, side effectsinclude, without limitation, injection site reactions, liver functiontest abnormalities, renal function abnormalities, liver toxicity, renaltoxicity, central nervous system abnormalities, and myopathies.

“Single-stranded oligonucleotide” means an oligonucleotide which is nothybridized to a complementary strand.

“Sites,” as used herein, are defined as unique nucleobase positionswithin a target nucleic acid.

“Specifically hybridizable” or “specifically hybridizes” refers to anantisense compound having a sufficient degree of complementarity betweenan antisense oligonucleotide and a target nucleic acid to induce adesired effect, while exhibiting minimal or no effects on non-targetnucleic acids under conditions in which specific binding is desired,i.e., under physiological conditions in the case of in vivo assays andtherapeutic treatments.

“Stringent hybridization conditions” or “stringent conditions” refer toconditions under which an oligomeric compound will hybridize to itstarget sequence, but to a minimal number of other sequences.

“Subject” means a human or non-human animal selected for treatment ortherapy.

“Target” refers to a protein, the modulation of which is desired.

“Target gene” refers to a gene encoding a target.

“Targeting” or “targeted” means the process of design and selection ofan antisense compound that will specifically hybridize to a targetnucleic acid and induce a desired effect.

“Target nucleic acid,” “target RNA,” and “target RNA transcript” and“nucleic acid target” all mean a nucleic acid capable of being targetedby antisense compounds.

“Target region” means a portion of a target nucleic acid to which one ormore antisense compounds is targeted.

“Target segment” means the sequence of nucleotides of a target nucleicacid to which an antisense compound is targeted. “5′ target site” refersto the 5′-most nucleotide of a target segment. “3′ target site” refersto the 3′-most nucleotide of a target segment.

“Therapeutically effective amount” means an amount of a pharmaceuticalagent that provides a therapeutic benefit to an individual.

“Treat” or “treating” or “treatment” refers to administering acomposition to effect an improvement of the disease or condition.

“Unmodified nucleobases” mean the purine bases adenine (A) and guanine(G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

“Unmodified nucleotide” means a nucleotide composed of naturallyoccurring nucleobases, sugar moieties, and internucleoside linkages. Incertain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e.β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).

“Upstream” refers to the relative direction toward the 5′ end orN-terminal end of a nucleic acid.

“Wing segment” means a plurality of nucleosides modified to impart to anoligonucleotide properties such as enhanced inhibitory activity,increased binding affinity for a target nucleic acid, or resistance todegradation by in vivo nucleases.

Certain Embodiments

Certain embodiments provide compounds, compositions, and methods forinhibiting plasma prekallikrein (PKK) mRNA and protein expression.Certain embodiments provide compounds, compositions, and methods fordecreasing PKK mRNA and protein levels.

Certain embodiments provide antisense compounds targeted to a plasmaprekallikrein (PKK) nucleic acid. In certain embodiments, the PKKnucleic acid is the sequence set forth in GENBANK Accession No.NM_000892.3 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No.DC412984.1 (incorporated herein as SEQ ID NO: 2), GENBANK Accession No.CN265612.1 (incorporated herein as SEQ ID NO: 3), GENBANK Accession No.AK297672.1 (incorporated herein as SEQ ID NO: 4), GENBANK Accession No.DC413312.1 (incorporated herein as SEQ ID NO: 5), GENBANK Accession No.AV688858.2 (incorporated herein as SEQ ID NO: 6), GENBANK Accession No.CD652077.1 (incorporated herein as SEQ ID NO: 7), GENBANK Accession No.BC143911.1 (incorporated herein as SEQ ID NO: 8), GENBANK Accession No.CB162532.1 (incorporated herein as SEQ ID NO: 9), GENBANK Accession No.NT_016354.19 truncated from nucleobases 111693001 to Ser. No. 11/730,000(incorporated herein as SEQ ID NO: 10), GENBANK Accession No.NM_008455.2 (incorporated herein as SEQ ID NO: 11), GENBANK AccessionNo. BB598673.1 (incorporated herein as SEQ ID NO: 12), GENBANK AccessionNo. NT_039460.7 truncated from nucleobases 6114001 to U.S. Pat. No.6,144,000 (incorporated herein as SEQ ID NO: 13), GENBANK Accession No.NM_012725.2 (incorporated herein as SEQ ID NO: 14), GENBANK AccessionNo. NW_047473.1 truncated from nucleobases 10952001 to Ser. No.10/982,000 (incorporated herein as SEQ ID NO: 15), GENBANK Accession No.XM_002804276.1 (incorporated herein as SEQ ID NO: 17), and GENBANKAccession No. NW_001118167.1 truncated from nucleobases 2358000 to U.S.Pat. No. 2,391,000 (incorporated herein as SEQ ID NO: 18).

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or at least 20 consecutivenucleobases of any of the nucleobase sequences of SEQ ID NOs: 30-2226.

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or at least 20 consecutivenucleobases of the nucleobase sequence of SEQ ID NO: 570.

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or at least 20 consecutivenucleobases of the nucleobase sequence of SEQ ID NO: 705.

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, or atleast 16 consecutive nucleobases of the nucleobase sequence of SEQ IDNO: 1666.

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 20 linked nucleosides and having thenucleobase sequence of SEQ ID NO: 570.

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 20 linked nucleosides and having thenucleobase sequence of SEQ ID NO: 705.

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 16 linked nucleosides and having thenucleobase sequence of SEQ ID NO: 1666.

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, or atleast 16 consecutive nucleobases of any of the nucleobase sequences ofSEQ ID NOs: 62, 72, 103, 213, 312, 334-339, 344, 345, 346, 348, 349,351, 369, 373, 381, 382, 383, 385, 387-391, 399, 411, 412, 414, 416,444, 446-449, 452, 453, 454, 459, 460, 462-472, 473, 476, 477, 479, 480,481, 484, 489-495, 497, 500, 504, 506, 522, 526, 535, 558, 559, 560,564, 566, 568-571, 573, 576, 577, 578, 587, 595, 597-604, 607, 608, 610,613, 615, 618, 619, 622, 623, 624, 633, 635, 636, 638, 639, 640, 642,643, 645, 652, 655-658, 660, 661, 670, 674-679, 684, 685, 698, 704, 705,707, 708, 713, 716, 717, 728, 734, 736, 767, 768, 776, 797, 798, 800,802, 810, 815, 876, 880, 882, 883, 886, 891, 901-905, 908-911, 922, 923,924, 931, 942, 950-957, 972, 974, 978, 979, 980, 987-991, 1005,1017-1021, 1025, 1026, 1029, 1030, 1032, 1034, 1035, 1037, 1040, 1041,1045, 1046, 1051, 1054, 1059, 1060, 1061, 1064, 1065, 1066, 1075, 1076,1087, 1089, 1111, 1114, 1116, 1117, 1125, 1133, 1153, 1169, 1177, 1181,1182, 1187, 1196, 1200, 1214, 1222, 1267, 1276, 1277, 1285, 1286, 1289,1290, 1291, 1303, 1367, 1389, 1393, 1398-1401, 1406, 1407, 1408, 1411,1419-1422, 1426, 1430, 1431, 1432, 1434-1437, 1439, 1440, 1443, 1444,1451, 1452, 1471, 1516, 1527, 1535, 1537, 1538, 1539, 1540, 1541, 1563,1564, 1567, 1568, 1616, 1617, 1623, 1629, 1664, 1665, 1666, 1679, 1687,1734, 1804, 1876, 1886, 1915, 2008, 2018, 2100, 2101, 2115, and 2116. Incertain embodiments, the modified oligonucleotide achieves at least 80%mRNA inhibition of PKK.

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, or atleast 16 consecutive nucleobases of any of the nucleobase sequences ofSEQ ID NOs: 62, 72, 103, 213, 334-339, 344, 346, 348, 349, 351, 381,382, 383, 385, 389, 390, 391, 446, 448, 452, 453, 454, 466-473, 476,481, 484, 491, 492, 494, 495, 497, 504, 526, 558, 559, 566, 568-571,576, 578, 587, 595, 597, 598, 600-604, 607, 610, 613, 618, 619, 624,635, 638, 639, 645, 652, 656, 657, 658, 660, 674, 675, 676, 684, 698,704, 705, 707, 713, 716, 768, 876, 880, 901-905, 908-911, 922, 923, 924,931, 942, 951, 954-957, 972, 974, 978, 979, 987, 988, 990, 1005, 1019,1020, 1021, 1025, 1032, 1037, 1040, 1041, 1045, 1054, 1059, 1060, 1061,1064, 1065, 1066, 1075, 1111, 1116, 1117, 1125, 1133, 1153, 1169, 1177,1200, 1222, 1267, 1285, 1290, 1291, 1303, 1367, 1398, 1399, 1401, 1406,1408, 1411, 1419, 1420, 1421, 1426, 1430, 1431, 1432, 1434-1437, 1440,1443, 1444, 1451, 1537-1540, 1563, 1616, 1679, 1687, 1804, 2008, 2101,2115, and 2116. In certain embodiments, the modified oligonucleotideachieves at least 85% mRNA inhibition of PKK.

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, or atleast 16 consecutive nucleobases of any of the nucleobase sequences ofSEQ ID NOs: 334, 346, 351, 382, 390, 391, 446, 448, 452, 453, 468, 469,470, 471, 472, 476, 481, 491, 495, 504, 558, 566, 568, 570, 571, 578,587, 597, 598, 600, 604, 613, 635, 638, 645, 656, 658, 660, 674, 675,684, 704, 705, 880, 901-905, 909, 922, 931, 951, 954, 956, 990, 1005,1020, 1032, 1037, 1040, 1041, 1045, 1054, 1075, 1111, 1125, 1133, 1153,1200, 1267, 1291, 1303, 1398, 1399, 1401, 1406, 1420, 1426, 1430, 1431,1434, 1435, 1436, 1440, 1443, 1451, 1537-1540, 2115, and 2116. Incertain embodiments, the modified oligonucleotide achieves at least 90%mRNA inhibition of PKK.

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, or atleast 16 consecutive nucleobases of any of the nucleobase sequences ofSEQ ID NOs: 334, 391, 448, 468, 469, 568, 570, 598, 635, 658, 674, 684,705, 901, 903, 904, 922, 990, 1267, 1291, 1420, 1430, 1431, 1434, 1435,1436, 1537, 1538, and 1540. In certain embodiments, the modifiedoligonucleotide achieves at least 95% mRNA inhibition of PKK.

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, or atleast 16 consecutive nucleobases of any of the nucleobase sequences ofSEQ ID NOs: 334, 338, 346, 349, 382, 383, 390, 448, 452, 453, 454, 495,526, 559, 570, 587, 598, 635, 660, 705, 901, 903, 904, 908, 923, 931,955, 974, 988, 990, 1020, 1039, 1040, 1111, 1117, 1267, 1291, 1349,1352, 1367, 1389, 1393, 1399, 1401, 1408, 1411, 1426, 1499, 1516, 1535,1544, 1548, 1563, 1564, 1568, 1569, 1598, 1616, 1617, 1623, 1624, 1643,1661, 1665, 1666, 1673, 1679, 1695, 1720, 1804, 1817, 1876, 1881, 1886,1940, 1947, 2008, 2018, 2019, 2031, 2044, 2100, 2101, 2115, and 2116. Incertain embodiments, the modified oligonucleotide achieves an IC₅₀ (μM)of 0.4 or less.

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, or atleast 16 consecutive nucleobases of any of the nucleobase sequences ofSEQ ID NOs: 334, 346, 349, 382, 453, 454, 495, 526, 570, 587, 598, 635,660, 901, 903, 904, 931, 955, 990, 1020, 1111, 1267, 1349, 1352, 1367,1389, 1399, 1408, 1411, 1426, 1516, 1535, 1544, 1548, 1563, 1564, 1568,1569, 1598, 1616, 1617, 1623, 1643, 1661, 1665, 1666, 1673, 1695, 1804,1876, 1881, 2019, 2044, 2100, 2101, 2115, and 2116. In certainembodiments, the modified oligonucleotide achieves an IC₅₀ (IM) of 0.3or less.

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, or atleast 16 consecutive nucleobases of any of the nucleobase sequences ofSEQ ID NOs: 334, 346, 382, 453, 495, 526, 570, 587, 598, 635, 901, 904,931, 955, 1020, 1111, 1349, 1352, 1389, 1426, 1516, 1535, 1544, 1548,1564, 1569, 1598, 1616, 1617, 1665, 1666, 1804, 1876, 1881, 2019, 2044,2101, and 2116. In certain embodiments, the modified oligonucleotideachieves an IC₅₀ (IM) of 0.2 or less.

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, or atleast 16 consecutive nucleobases of any of the nucleobase sequences ofSEQ ID NOs: 334, 495, 587, 598, 635, 1349, 1352, 1389, 1516, 1544, 1548,1569, 1598, 1617, 1665, 1666, 1804, 1881, and 2019. In certainembodiments, the modified oligonucleotide achieves an IC₅₀ (IM) of lessthan 0.2.

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and comprisinga nucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or at least 20 consecutivenucleobases complementary to an equal length portion of nucleobases27427-27466 of SEQ ID NO: 10.

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and comprisinga nucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or at least 20 consecutivenucleobases complementary to an equal length portion of nucleobases33183-33242 of SEQ ID NO: 10.

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and comprisinga nucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or at least 20 consecutivenucleobases complementary to an equal length portion of nucleobases30570-30610 of SEQ ID NO: 10.

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and comprisinga nucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or at least 20 consecutivenucleobases complementary to an equal length portion of nucleobases27427-27520 of SEQ ID NO: 10.

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and comprisinga nucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or at least 20 consecutivenucleobases complementary to an equal length portion of nucleobases33085-33247 of SEQ ID NO: 10.

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and comprisinga nucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or at least 20 consecutivenucleobases complementary to an equal length portion of nucleobases30475-30639 of SEQ ID NO: 10.

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and comprisinga nucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or at least 20 consecutivenucleobases complementary to an equal length portion of nucleobases27362-27524 of SEQ ID NO: 10.

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and comprisinga nucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or at least 20 consecutivenucleobases complementary to an equal length portion of nucleobases33101-33240 of SEQ ID NO: 10.

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and comprisinga nucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or at least 20 consecutivenucleobases complementary to an equal length portion of nucleobases30463-30638 of SEQ ID NO: 10.

Certain embodiments provide compounds, comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and comprisinga nucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or at least 20 consecutivenucleobases complementary to an equal length portion of exon 9, exon 12,or exon 14 of a PKK nucleic acid.

In certain embodiments the nucleobase sequence of the modifiedoligonucleotide is at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% complementary to SEQ ID NO: 10.

In certain embodiments, the compound consists of a single-strandedmodified oligonucleotide.

In certain embodiments, at least one internucleoside linkage of themodified oligonucleotide is a modified intemucleoside linkage.

In certain embodiments, at least one modified intemucleoside linkage ofthe modified oligonucleotide is a phosphorothioate intemucleosidelinkage.

In certain embodiments, each intemucleoside linkage of the modifiedoligonucleotide is a phosphorothioate linkage.

In certain embodiments, at least one nucleoside of the modifiedoligonucleotide comprises a modified nucleobase.

In certain embodiments, the modified nucleobase is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide comprises at leastone modified sugar.

In certain embodiments, the modified sugar is a 2′ modified sugar, aBNA, or a THP.

In certain embodiments, the modified sugar is any of a2′-O-methoxyethyl, 2′-O-methyl, a constrained ethyl, a LNA, or a3′-fluoro-HNA.

In certain embodiments, the compound comprises at least one2′-O-methoxyethyl nucleoside, 2′-O-methyl nucleoside, constrained ethylnucleoside, LNA nucleoside, or 3′-fluoro-HNA nucleoside.

In certain embodiments, the modified oligonucleotide comprises:

a gap segment consisting of 10 linked deoxynucleosides;

a 5′ wing segment consisting of 5 linked nucleosides; and

a 3′ wing segment consisting of 5 linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment and wherein each nucleoside of each wing segmentcomprises a modified sugar.

In certain embodiments, the modified oligonucleotide consists of 20linked nucleosides.

In certain embodiments, the modified oligonucleotide consists of 19linked nucleosides.

In certain embodiments, the modified oligonucleotide consists of 18linked nucleosides.

Certain embodiments provide compounds consisting of a modifiedoligonucleotide according to the following formula: Tes Ges mCes Aes AesGds Tds mCds Tds mCds Tds Tds Gds Gds mCds Aes Aes Aes mCes Ae; wherein,

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

Certain embodiments provide compounds consisting of a modifiedoligonucleotide according to the following formula: mCes mCes mCes mCesmCes Tds Tds mCds Tds Tds Tds Ads Tds Ads Gds mCes mCes Aes Ges mCe;wherein,

A=an adenine,

mC=a 5′-methylcytosine;

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

Certain embodiments provide compounds consisting of a modifiedoligonucleotide according to the following formula: mCes Ges Aks Tds AdsTds mCds Ads Tds Gds Ads Tds Tds mCks mCks mCe; wherein,

A=an adenine,

mC=a 5′-methylcytosine;

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

k=a cEt modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

Certain embodiments provide compounds according to the followingformula:

Certain embodiments provide compounds according to the followingformula:

Certain embodiments provide compounds according to the followingformula:

Certain embodiments provide compositions comprising the compound of anypreceding claim or salt thereof and at least one of a pharmaceuticallyacceptable carrier or diluent.

Certain embodiments provide methods comprising administering to ananimal the compound or composition of any preceding claim.

In certain embodiments, the animal is a human.

In certain embodiments, administering the compound prevents, treats, orameliorates a PKK associated disease, disorder or condition.

In certain embodiments, the PKK associated disease, disorder orcondition is a hereditary angioedema (HAE), edema, angioedema, swelling,angioedema of the lids, ocular edema, macular edema, cerebral edema,thrombosis, embolism, thromboembolism, deep vein thrombosis, pulmonaryembolism, myocardial infarction, stroke, or infarct.

Certain embodiments provide use of the compound or composition of anypreceding claim for the manufacture of a medicament for treating aninflammatory disease or a thromboembolic disease.

Antisense Compounds

Oligomeric compounds include, but are not limited to, oligonucleotides,oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics,antisense compounds, antisense oligonucleotides, and siRNAs. Anoligomeric compound may be “antisense” to a target nucleic acid, meaningthat is capable of undergoing hybridization to a target nucleic acidthrough hydrogen bonding.

In certain embodiments, an antisense compound has a nucleobase sequencethat, when written in the 5′ to 3′ direction, comprises the reversecomplement of the target segment of a target nucleic acid to which it istargeted. In certain such embodiments, an antisense oligonucleotide hasa nucleobase sequence that, when written in the 5′ to 3′ direction,comprises the reverse complement of the target segment of a targetnucleic acid to which it is targeted.

In certain embodiments, an antisense compound targeted to a PKK nucleicacid is 12 to 30 subunits in length. In certain embodiments, anantisense compound targeted to PKK nucleic acid is 12 to 25 subunits inlength. In certain embodiments, an antisense compound targeted to PKKnucleic acid is 12 to 22 subunits in length. In certain embodiments, anantisense compound targeted to PKK nucleic acid is 14 to 20 subunits inlength. In certain embodiments, an antisense compound targeted to PKKnucleic acid is 15 to 25 subunits in length. In certain embodiments, anantisense compound targeted to PKK nucleic acid is 18 to 22 subunits inlength. In certain embodiments, an antisense compound targeted to PKKnucleic acid is 19 to 21 subunits in length. In certain embodiments, theantisense compound is 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30,14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18to 30, 18 to 50, 19 to 30, 19 to 50, or 20 to 30 linked subunits inlength.

In certain embodiments, an antisense compound targeted to a PKK nucleicacid is 12 subunits in length. In certain embodiments, an antisensecompound targeted to a PKK nucleic acid is 13 subunits in length. Incertain embodiments, an antisense compound targeted to a PKK nucleicacid is 14 subunits in length. In certain embodiments, an antisensecompound targeted to a PKK nucleic acid is 15 subunits in length. Incertain embodiments, an antisense compound targeted to a PKK nucleicacid is 16 subunits in length. In certain embodiments, an antisensecompound targeted to a PKK nucleic acid is 17 subunits in length. Incertain embodiments, an antisense compound targeted to a PKK nucleicacid is 18 subunits in length. In certain embodiments, an antisensecompound targeted to a PKK nucleic acid is 19 subunits in length. Incertain embodiments, an antisense compound targeted to a PKK nucleicacid is 20 subunits in length. In certain embodiments, an antisensecompound targeted to a PKK nucleic acid is 21 subunits in length. Incertain embodiments, an antisense compound targeted to a PKK nucleicacid is 22 subunits in length. In certain embodiments, an antisensecompound targeted to a PKK nucleic acid is 23 subunits in length. Incertain embodiments, an antisense compound targeted to a PKK nucleicacid is 24 subunits in length. In certain embodiments, an antisensecompound targeted to a PKK nucleic acid is 25 subunits in length. Incertain embodiments, an antisense compound targeted to a PKK nucleicacid is 26 subunits in length. In certain embodiments, an antisensecompound targeted to a PKK nucleic acid is 27 subunits in length. Incertain embodiments, an antisense compound targeted to a PKK nucleicacid is 28 subunits in length. In certain embodiments, an antisensecompound targeted to a PKK nucleic acid is 29 subunits in length. Incertain embodiments, an antisense compound targeted to a PKK nucleicacid is 30 subunits in length. In certain embodiments, the antisensecompound targeted to a PKK nucleic acid is 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a rangedefined by any two of the above values. In certain embodiments theantisense compound is an antisense oligonucleotide, and the linkedsubunits are nucleosides.

In certain embodiments antisense oligonucleotides targeted to a PKKnucleic acid may be shortened or truncated. For example, a singlesubunit may be deleted from the 5′ end (5′ truncation), or alternativelyfrom the 3′ end (3′ truncation). A shortened or truncated antisensecompound targeted to a PKK nucleic acid may have two subunits deletedfrom the 5′ end, or alternatively may have two subunits deleted from the3′ end, of the antisense compound. Alternatively, the deletednucleosides may be dispersed throughout the antisense compound, forexample, in an antisense compound having one nucleoside deleted from the5′ end and one nucleoside deleted from the 3′ end.

When a single additional subunit is present in a lengthened antisensecompound, the additional subunit may be located at the 5′ or 3′ end ofthe antisense compound. When two or more additional subunits arepresent, the added subunits may be adjacent to each other, for example,in an antisense compound having two subunits added to the 5′ end (5′addition), or alternatively to the 3′ end (3′ addition), of theantisense compound. Alternatively, the added subunits may be dispersedthroughout the antisense compound, for example, in an antisense compoundhaving one subunit added to the 5′ end and one subunit added to the 3′end.

It is possible to increase or decrease the length of an antisensecompound, such as an antisense oligonucleotide, and/or introducemismatch bases without eliminating activity. For example, in Woolf etal. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series ofantisense oligonucleotides 13-25 nucleobases in length were tested fortheir ability to induce cleavage of a target RNA in an oocyte injectionmodel. Antisense oligonucleotides 25 nucleobases in length with 8 or 11mismatch bases near the ends of the antisense oligonucleotides were ableto direct specific cleavage of the target mRNA, albeit to a lesserextent than the antisense oligonucleotides that contained no mismatches.Similarly, target specific cleavage was achieved using 13 nucleobaseantisense oligonucleotides, including those with 1 or 3 mismatches.

Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001)demonstrated the ability of an oligonucleotide having 100%complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xLmRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and invivo. Furthermore, this oligonucleotide demonstrated potent anti-tumoractivity in vivo.

Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a seriesof tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42nucleobase antisense oligonucleotides comprised of the sequence of twoor three of the tandem antisense oligonucleotides, respectively, fortheir ability to arrest translation of human DHFR in a rabbitreticulocyte assay. Each of the three 14 nucleobase antisenseoligonucleotides alone was able to inhibit translation, albeit at a moremodest level than the 28 or 42 nucleobase antisense oligonucleotides.

Antisense Compound Motifs

In certain embodiments, antisense compounds targeted to a PKK nucleicacid have chemically modified subunits arranged in patterns, or motifs,to confer to the antisense compounds properties such as enhancedinhibitory activity, increased binding affinity for a target nucleicacid, or resistance to degradation by in vivo nucleases.

Chimeric antisense compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, increased binding affinity for the targetnucleic acid, and/or increased inhibitory activity. A second region of achimeric antisense compound may optionally serve as a substrate for thecellular endonuclease RNase H, which cleaves the RNA strand of anRNA:DNA duplex.

Antisense compounds having a gapmer motif are considered chimericantisense compounds. In a gapmer an internal region having a pluralityof nucleotides that supports RNaseH cleavage is positioned betweenexternal regions having a plurality of nucleotides that are chemicallydistinct from the nucleosides of the internal region. In the case of anantisense oligonucleotide having a gapmer motif, the gap segmentgenerally serves as the substrate for endonuclease cleavage, while thewing segments comprise modified nucleosides. In certain embodiments, theregions of a gapmer are differentiated by the types of sugar moietiescomprising each distinct region. The types of sugar moieties that areused to differentiate the regions of a gapmer may in some embodimentsinclude β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modifiednucleosides (such 2′-modified nucleosides may include 2′-MOE, and2′-O—CH₃, among others), and bicyclic sugar modified nucleosides (suchbicyclic sugar modified nucleosides may include those having a4′-(CH₂)n-O-2′ bridge, where n=1 or n=2 and 4′-CH₂—O—CH₂-2′). In certainembodiments, wings may include several modified sugar moieties,including, for example 2′-MOE. In certain embodiments, wings may includeseveral modified and unmodified sugar moieties. In certain embodiments,wings may include various combinations of 2′-MOE nucleosides and2′-deoxynucleosides.

Each distinct region may comprise uniform sugar moieties, variant, oralternating sugar moieties. The wing-gap-wing motif is frequentlydescribed as “X-Y-Z”, where “X” represents the length of the 5′ wing,“Y” represents the length of the gap, and “Z” represents the length ofthe 3′ wing. “X” and “Z” may comprise uniform, variant, or alternatingsugar moieties. In certain embodiments, “X” and “Y” may include one ormore 2′-deoxynucleosides. “Y” may comprise 2′-deoxynucleosides. As usedherein, a gapmer described as “X-Y-Z” has a configuration such that thegap is positioned immediately adjacent to each of the 5′ wing and the 3′wing. Thus, no intervening nucleotides exist between the 5′ wing andgap, or the gap and the 3′ wing. Any of the antisense compoundsdescribed herein can have a gapmer motif. In certain embodiments, “X”and “Z” are the same; in other embodiments they are different.

In certain embodiments, gapmers provided herein include, for example20-mers having a motif of 5-10-5.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

Nucleotide sequences that encode human plasma prekallikrein (PKK)include, without limitation, the following: GENBANK Accession No.NM_000892.3 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No.DC412984.1 (incorporated herein as SEQ ID NO: 2), GENBANK Accession No.CN265612.1 (incorporated herein as SEQ ID NO: 3), GENBANK Accession No.AK297672.1 (incorporated herein as SEQ ID NO: 4), GENBANK Accession No.DC413312.1 (incorporated herein as SEQ ID NO: 5), GENBANK Accession No.AV688858.2 (incorporated herein as SEQ ID NO: 6), GENBANK Accession No.CD652077.1 (incorporated herein as SEQ ID NO: 7), GENBANK Accession No.BC143911.1 (incorporated herein as SEQ ID NO: 8), GENBANK Accession No.CB162532.1 (incorporated herein as SEQ ID NO: 9), GENBANK Accession No.NT 016354.19 truncated from nucleobases 111693001 to Ser. No. 11/730,000(incorporated herein as SEQ ID NO: 10), GENBANK Accession No.NM_008455.2 (incorporated herein as SEQ ID NO: 11), GENBANK AccessionNo. BB598673.1 (incorporated herein as SEQ ID NO: 12), GENBANK AccessionNo. NT_039460.7 truncated from nucleobases 6114001 to U.S. Pat. No.6,144,000 (incorporated herein as SEQ ID NO: 13), GENBANK Accession No.NM_012725.2 (incorporated herein as SEQ ID NO: 14), GENBANK AccessionNo. NW_047473.1 truncated from nucleobases 10952001 to Ser. No.10/982,000 (incorporated herein as SEQ ID NO: 15), GENBANK Accession No.XM_002804276.1 (incorporated herein as SEQ ID NO: 17), and GENBANKAccession No. NW_001118167.1 truncated from nucleobases 2358000 to U.S.Pat. No. 2,391,000 (incorporated herein as SEQ ID NO: 18).

It is understood that the sequence set forth in each SEQ ID NO in theExamples contained herein is independent of any modification to a sugarmoiety, an internucleoside linkage, or a nucleobase. As such, antisensecompounds defined by a SEQ ID NO may comprise, independently, one ormore modifications to a sugar moiety, an internucleoside linkage, or anucleobase. Antisense compounds described by Isis Number (Isis No)indicate a combination of nucleobase sequence and motif.

In certain embodiments, a target region is a structurally defined regionof the target nucleic acid. For example, a target region may encompass a3′ UTR, a 5′ UTR, an exon, an intron, an exon/intron junction, a codingregion, a translation initiation region, translation termination region,or other defined nucleic acid region. The structurally defined regionsfor PKK can be obtained by accession number from sequence databases suchas NCBI and such information is incorporated herein by reference. Incertain embodiments, a target region may encompass the sequence from a5′ target site of one target segment within the target region to a 3′target site of another target segment within the same target region.

Targeting includes determination of at least one target segment to whichan antisense compound hybridizes, such that a desired effect occurs. Incertain embodiments, the desired effect is a reduction in mRNA targetnucleic acid levels. In certain embodiments, the desired effect isreduction of levels of protein encoded by the target nucleic acid or aphenotypic change associated with the target nucleic acid.

A target region may contain one or more target segments. Multiple targetsegments within a target region may be overlapping. Alternatively, theymay be non-overlapping. In certain embodiments, target segments within atarget region are separated by no more than about 300 nucleotides. Incertain embodiments, target segments within a target region areseparated by a number of nucleotides that is, is about, is no more than,is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30,20, or 10 nucleotides on the target nucleic acid, or is a range definedby any two of the preceeding values. In certain embodiments, targetsegments within a target region are separated by no more than, or nomore than about, 5 nucleotides on the target nucleic acid. In certainembodiments, target segments are contiguous. Contemplated are targetregions defined by a range having a starting nucleic acid that is any ofthe 5′ target sites or 3′ target sites listed herein.

Suitable target segments may be found within a 5′ UTR, a coding region,a 3′ UTR, an intron, an exon, or an exon/intron junction. Targetsegments containing a start codon or a stop codon are also suitabletarget segments. A suitable target segment may specifically exclude acertain structurally defined region such as the start codon or stopcodon.

The determination of suitable target segments may include a comparisonof the sequence of a target nucleic acid to other sequences throughoutthe genome. For example, the BLAST algorithm may be used to identifyregions of similarity amongst different nucleic acids. This comparisoncan prevent the selection of antisense compound sequences that mayhybridize in a non-specific manner to sequences other than a selectedtarget nucleic acid (i.e., non-target or off-target sequences).

There may be variation in activity (e.g., as defined by percentreduction of target nucleic acid levels) of the antisense compoundswithin an active target region. In certain embodiments, reductions inPKK mRNA levels are indicative of inhibition of PKK expression.Reductions in levels of a PKK protein are also indicative of inhibitionof target mRNA expression. Further, phenotypic changes are indicative ofinhibition of PKK expression. For example, reduced or preventedinflammation can be indicative of inhibition of PKK expression. Inanother example, reduced or prevented edema/swelling can be indicativeof inhibition of PKK expression. In another example, reduced orprevented vascular permeability can be indicative of inhibition of PKKexpression. In another example, reduced or prevented vascular leakagecan be indicative of inhibition of PKK expression. In certainembodiments, vascular permeability is measured by quantification of adye, such as Evans Blue.

Hybridization

In some embodiments, hybridization occurs between an antisense compounddisclosed herein and a target nucleic acid. The most common mechanism ofhybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteenor reversed Hoogsteen hydrogen bonding) between complementarynucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Stringent conditionsare sequence-dependent and are determined by the nature and compositionof the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizableto a target nucleic acid are well known in the art. In certainembodiments, the antisense compounds provided herein are specificallyhybridizable with a target nucleic acid.

Complementarity

An antisense compound and a target nucleic acid are complementary toeach other when a sufficient number of nucleobases of the antisensecompound can hydrogen bond with the corresponding nucleobases of thetarget nucleic acid, such that a desired effect will occur (e.g.,antisense inhibition of a target nucleic acid, such as a PKK nucleicacid).

Non-complementary nucleobases between an antisense compound and a PKKnucleic acid may be tolerated provided that the antisense compoundremains able to specifically hybridize to a target nucleic acid.Moreover, an antisense compound may hybridize over one or more segmentsof a PKK nucleic acid such that intervening or adjacent segments are notinvolved in the hybridization event (e.g., a loop structure, mismatch orhairpin structure).

In certain embodiments, the antisense compounds provided herein, or aspecified portion thereof, are, or are at least, 70%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% complementary to an PKK nucleic acid, a targetregion, target segment, or specified portion thereof. Percentcomplementarity of an antisense compound with a target nucleic acid canbe determined using routine methods.

For example, an antisense compound in which 18 of 20 nucleobases of theantisense compound are complementary to a target region, and wouldtherefore specifically hybridize, would represent 90 percentcomplementarity. In this example, the remaining noncomplementarynucleobases may be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an antisense compound which is 18 nucleobases inlength having four noncomplementary nucleobases which are flanked by tworegions of complete complementarity with the target nucleic acid wouldhave 77.8% overall complementarity with the target nucleic acid andwould thus fall within the scope of the present invention. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden,Genome Res., 1997, 7, 649 656). Percent homology, sequence identity orcomplementarity, can be determined by, for example, the Gap program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, Madison Wis.), using defaultsettings, which uses the algorithm of Smith and Waterman (Adv. Appl.Math., 1981, 2, 482 489).

In certain embodiments, the antisense compounds provided herein, orspecified portions thereof, are fully complementary (i.e. 100%complementary) to a target nucleic acid, or specified portion thereof.For example, an antisense compound may be fully complementary to aplasma prekallikrein nucleic acid, or a target region, or a targetsegment or target sequence thereof. As used herein, “fullycomplementary” means each nucleobase of an antisense compound is capableof precise base pairing with the corresponding nucleobases of a targetnucleic acid. For example, a 20 nucleobase antisense compound is fullycomplementary to a target sequence that is 400 nucleobases long, so longas there is a corresponding 20 nucleobase portion of the target nucleicacid that is fully complementary to the antisense compound. Fullycomplementary can also be used in reference to a specified portion ofthe first and/or the second nucleic acid. For example, a 20 nucleobaseportion of a 30 nucleobase antisense compound can be “fullycomplementary” to a target sequence that is 400 nucleobases long. The 20nucleobase portion of the 30 nucleobase oligonucleotide is fullycomplementary to the target sequence if the target sequence has acorresponding 20 nucleobase portion wherein each nucleobase iscomplementary to the 20 nucleobase portion of the antisense compound. Atthe same time, the entire 30 nucleobase antisense compound may or maynot be fully complementary to the target sequence, depending on whetherthe remaining 10 nucleobases of the antisense compound are alsocomplementary to the target sequence.

The location of a non-complementary nucleobase may be at the 5′ end or3′ end of the antisense compound. Alternatively, the non-complementarynucleobase or nucleobases may be at an internal position of theantisense compound. When two or more non-complementary nucleobases arepresent, they may be contiguous (i.e. linked) or non-contiguous. In oneembodiment, a non-complementary nucleobase is located in the wingsegment of a gapmer antisense oligonucleotide.

In certain embodiments, antisense compounds that are, or are up to 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise nomore than 4, no more than 3, no more than 2, or no more than 1non-complementary nucleobase(s) relative to a target nucleic acid orspecified portion thereof.

In certain embodiments, antisense compounds that are, or are up to 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30 nucleobases in length comprise no more than 6, no more than 5, nomore than 4, no more than 3, no more than 2, or no more than 1non-complementary nucleobase(s) relative to a target nucleic acid orspecified portion thereof.

The antisense compounds provided also include those which arecomplementary to a portion of a target nucleic acid. As used herein,“portion” refers to a defined number of contiguous (i.e. linked)nucleobases within a region or segment of a target nucleic acid. A“portion” can also refer to a defined number of contiguous nucleobasesof an antisense compound. In certain embodiments, the antisensecompounds, are complementary to at least an 8 nucleobase portion of atarget segment. In certain embodiments, the antisense compounds arecomplementary to at least a 9 nucleobase portion of a target segment. Incertain embodiments, the antisense compounds are complementary to atleast a 10 nucleobase portion of a target segment. In certainembodiments, the antisense compounds are complementary to at least an 11nucleobase portion of a target segment. In certain embodiments, theantisense compounds are complementary to at least a 12 nucleobaseportion of a target segment. In certain embodiments, the antisensecompounds are complementary to at least a 13 nucleobase portion of atarget segment. In certain embodiments, the antisense compounds arecomplementary to at least a 14 nucleobase portion of a target segment.In certain embodiments, the antisense compounds are complementary to atleast a 15 nucleobase portion of a target segment. Also contemplated areantisense compounds that are complementary to at least a 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a targetsegment, or a range defined by any two of these values.

Identity

The antisense compounds provided herein may also have a defined percentidentity to a particular nucleotide sequence, SEQ ID NO, or compoundrepresented by a specific Isis number, or portion thereof. As usedherein, an antisense compound is identical to the sequence disclosedherein if it has the same nucleobase pairing ability. For example, a RNAwhich contains uracil in place of thymidine in a disclosed DNA sequencewould be considered identical to the DNA sequence since both uracil andthymidine pair with adenine. Shortened and lengthened versions of theantisense compounds described herein as well as compounds havingnon-identical bases relative to the antisense compounds provided hereinalso are contemplated. The non-identical bases may be adjacent to eachother or dispersed throughout the antisense compound. Percent identityof an antisense compound is calculated according to the number of basesthat have identical base pairing relative to the sequence to which it isbeing compared.

In certain embodiments, the antisense compounds, or portions thereof,are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to one or more of the antisense compounds or SEQ ID NOs, or aportion thereof, disclosed herein.

In certain embodiments, a portion of the antisense compound is comparedto an equal length portion of the target nucleic acid. In certainembodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, or 25 nucleobase portion is compared to an equal lengthportion of the target nucleic acid.

In certain embodiments, a portion of the antisense oligonucleotide iscompared to an equal length portion of the target nucleic acid. Incertain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equallength portion of the target nucleic acid.

Modifications

A nucleoside is a base-sugar combination. The nucleobase (also known asbase) portion of the nucleoside is normally a heterocyclic base moiety.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar.Oligonucleotides are formed through the covalent linkage of adjacentnucleosides to one another, to form a linear polymeric oligonucleotide.Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside linkages of theoligonucleotide.

Modifications to antisense compounds encompass substitutions or changesto internucleoside linkages, sugar moieties, or nucleobases. Modifiedantisense compounds are often preferred over native forms because ofdesirable properties such as, for example, enhanced cellular uptake,enhanced affinity for nucleic acid target, increased stability in thepresence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides may also be employed to increase thebinding affinity of a shortened or truncated antisense oligonucleotidefor its target nucleic acid. Consequently, comparable results can oftenbe obtained with shorter antisense compounds that have such chemicallymodified nucleosides.

Modified Internucleoside Linkages

The naturally occurring internucleoside linkage of RNA and DNA is a 3′to 5′ phosphodiester linkage. Antisense compounds having one or moremodified, i.e. non-naturally occurring, internucleoside linkages areoften selected over antisense compounds having naturally occurringinternucleoside linkages because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for target nucleicacids, and increased stability in the presence of nucleases.

Oligonucleotides having modified internucleoside linkages includeinternucleoside linkages that retain a phosphorus atom as well asinternucleoside linkages that do not have a phosphorus atom.Representative phosphorus containing internucleoside linkages include,but are not limited to, phosphodiesters, phosphotriesters,methylphosphonates, phosphoramidate, and phosphorothioates. Methods ofpreparation of phosphorous-containing and non-phosphorous-containinglinkages are well known.

In certain embodiments, antisense compounds targeted to a plasmaprekallikrein nucleic acid comprise one or more modified internucleosidelinkages. In certain embodiments, the modified intemucleoside linkagesare phosphorothioate linkages. In certain embodiments, eachinternucleoside linkage of an antisense compound is a phosphorothioateinternucleoside linkage.

Modified Sugar Moieties

Antisense compounds can optionally contain one or more nucleosideswherein the sugar group has been modified. Such sugar modifiednucleosides may impart enhanced nuclease stability, increased bindingaffinity, or some other beneficial biological property to the antisensecompounds. In certain embodiments, nucleosides comprise chemicallymodified ribofuranose ring moieties. Examples of chemically modifiedribofuranose rings include without limitation, addition of substitutentgroups (including 5′ and 2′ substituent groups, bridging of non-geminalring atoms to form bicyclic nucleic acids (BNA), replacement of theribosyl ring oxygen atom with S, N(R), or C(R₁)(R₂) (R, R₁ and R₂ areeach independently H, C₁-C₁₂ alkyl or a protecting group) andcombinations thereof. Examples of chemically modified sugars include2′-F-5′-methyl substituted nucleoside (see PCT International ApplicationWO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bissubstituted nucleosides) or replacement of the ribosyl ring oxygen atomwith S with further substitution at the 2′-position (see published U.S.Patent Application US2005-0130923, published on Jun. 16, 2005) oralternatively 5′-substitution of a BNA (see PCT InternationalApplication WO 2007/134181 Published on Nov. 22, 2007 wherein LNA issubstituted with for example a 5′-methyl or a 5′-vinyl group).

Examples of nucleosides having modified sugar moieties include withoutlimitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S,2′-F, 2′-OCH₃, 2′-OCH₂CH₃, 2′-OCH₂CH₂F and 2′-O(CH₂)₂OCH₃ substituentgroups. The substituent at the 2′ position can also be selected fromallyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, OCF₃, OCH₂F,O(CH₂)₂SCH₃, O(CH₂)₂—O—N(R_(m))(R_(n)), O—CH₂—C(═O)—N(R_(m))(R_(n)), andO—CH₂—C(═O)—N(R_(l))—(CH₂)₂—N(R_(m))(R_(n)), where each R_(l), R_(m) andR_(n) is, independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.

As used herein, “bicyclic nucleosides” refer to modified nucleosidescomprising a bicyclic sugar moiety. Examples of bicyclic nucleosidesinclude without limitation nucleosides comprising a bridge between the4′ and the 2′ ribosyl ring atoms. In certain embodiments, antisensecompounds provided herein include one or more bicyclic nucleosidescomprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclicnucleosides, include but are not limited to one of the formulas:4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′; 4′-(CH₂)₂—O-2′ (ENA);4′-CH(CH₃)—O-2′ (also referred to as constrained ethyl or cEt) and4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof see U.S. Pat. No. 7,399,845,issued on Jul. 15, 2008); 4′-C(CH₃)(CH₃)—O-2′ (and analogs thereof seePCT/US2008/068922 published as WO/2009/006478, published Jan. 8, 2009);4′-CH₂—N(OCH₃)-2′ (and analogs thereof see PCT/US2008/064591 publishedas WO/2008/150729, published Dec. 11, 2008); 4′-CH₂—O—N(CH₃)-2′ (seepublished U.S. Patent Application US2004-0171570, published Sep. 2,2004); 4′-CH₂—N(R)—O-2′, wherein R is H, C₁-C₁₂ alkyl, or a protectinggroup (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008);4′-CH₂—C(H)(CH₃)-2′ (see Chattopadhyaya et al., J. Org. Chem., 2009, 74,118-134); and 4′-CH₂—C(═CH₂)-2′ (and analogs thereof seePCT/US2008/066154 published as WO 2008/154401, published on Dec. 8,2008).

Further reports related to bicyclic nucleosides can also be found inpublished literature (see for example: Frieden et al., Nucleic AcidsResearch, 2003, 21, 6365-6372; Singh et al., Chem. Commun., 1998, 4,455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt etal., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al.,Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org.Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc.,2007, 129(26) 8362-8379; Elayadi et al., Curr. Opinion Invest. Drugs,2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; and Orum etal., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos.6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 7,034,133;7,053,207; 7,399,845; 7,547,684; and 7,696,345; U.S. Patent PublicationNo. US2008-0039618; US2009-0012281; US2007-0287831; US2004-0171570; U.S.patent application Ser. Nos. 12/129,154; 60/989,574; 61/026,995;61/026,998; 61/056,564; 61/086,231; 61/097,787; and 61/099,844;Published PCT International applications WO 1994/014226; WO 2004/106356;WO 2005/021570; WO 2007/134181; WO 2008/150729; WO 2008/154401; and WO2009/006478. Each of the foregoing bicyclic nucleosides can be preparedhaving one or more stereochemical sugar configurations including forexample α-L-ribofuranose and β-D-ribofuranose (see PCT internationalapplication PCT/DK98/00393, published on Mar. 25, 1999 as WO 99/14226).

In certain embodiments, bicyclic sugar moieties of BNA nucleosidesinclude, but are not limited to, compounds having at least one bridgebetween the 4′ and the 2′ position of the pentofuranosyl sugar moietywherein such bridges independently comprise 1 or from 2 to 4 linkedgroups independently selected from —[C(R_(a))(R_(b))]_(n)—,—C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═O)—, —C(═NR_(a))—, —C(═S)—, —O—,—Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃,COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), orsulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl ora protecting group.

In certain embodiments, the bridge of a bicyclic sugar moiety is—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—,—C(R_(a)R_(b))—N(R)—O— or —C(R_(a)R_(b))—O—N(R)—. In certainembodiments, the bridge is 4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′,4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R)-2′ and 4′-CH₂—N(R)—O-2′-wherein each R is, independently, H, a protecting group or C₁-C₁₂ alkyl.

In certain embodiments, bicyclic nucleosides are further defined byisomeric configuration. For example, a nucleoside comprising a 4′-2′methylene-oxy bridge, may be in the α-L configuration or in the β-Dconfiguration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) BNA's havebeen incorporated into antisense oligonucleotides that showed antisenseactivity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

In certain embodiments, bicyclic nucleosides include, but are notlimited to, (A) α-L-methyleneoxy (4′-CH₂—O-2′) BNA, (B) 3-D-methyleneoxy(4′-CH₂—O-2′) BNA, (C) ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) aminooxy(4′-CH₂—O—N(R)-2′) BNA, (E) oxyamino (4′-CH₂—N(R)—O-2′) BNA, and (F)methyl(methyleneoxy) (4′-CH(CH₃)—O-2′) BNA, (G) methylene-thio(4′-CH₂—S-2′) BNA, (H) methylene-amino (4′-CH₂—N(R)-2′) BNA, (I) methylcarbocyclic (4′-CH₂—CH(CH₃)-2′) BNA, (J) propylene carbocyclic(4′-(CH₂)₃-2′) BNA and (K) vinyl BNA as depicted below:

wherein Bx is the base moiety and R is independently H, a protectinggroup, C₁-C₁₂ alkyl or C₁-C₁₂ alkoxy.

In certain embodiments, bicyclic nucleosides are provided having FormulaI:

wherein:

Bx is a heterocyclic base moiety;

-Q_(a)-Q_(b)-Q_(c)- is —CH₂—N(R_(c))—CH₂—, —C(═O)—N(R_(c))—CH₂—,—CH₂—O—N(R_(c))—, —CH₂—N(R_(c))—O— or —N(R_(c))—O—CH₂;

R_(c) is C₁-C₁₂ alkyl or an amino protecting group; and

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium.

In certain embodiments, bicyclic nucleosides are provided having FormulaII:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

Z_(a) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, acyl,substituted acyl, substituted amide, thiol or substituted thio.

In one embodiment, each of the substituted groups is, independently,mono or poly substituted with substituent groups independently selectedfrom halogen, oxo, hydroxyl, OJ_(c), NJ_(c)J_(d), SJ_(c), N₃,OC(═X)J_(c), and NJ_(e)C(═X)NJ_(c)J_(d), wherein each J_(c), J_(d) andJ_(e) is, independently, H, C₁-C₆ alkyl, or substituted C₁-C₆ alkyl andX is O or NJ_(c).

In certain embodiments, bicyclic nucleosides are provided having FormulaIII:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

Z_(b) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl orsubstituted acyl (C(═O)—).

In certain embodiments, bicyclic nucleosides are provided having FormulaIV:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

R_(d) is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each q_(a), q_(b), q_(c) and q_(d) is, independently, H, halogen, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl, C₁-C₆ alkoxyl,substituted C₁-C₆ alkoxyl, acyl, substituted acyl, C₁-C₆ aminoalkyl orsubstituted C₁-C₆ aminoalkyl;

In certain embodiments, bicyclic nucleosides are provided having FormulaV:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

q_(a), q_(b), q_(e) and q_(f) are each, independently, hydrogen,halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl,C₁-C₁₂ alkoxy, substituted C₁-C₁₂ alkoxy, OJ_(j), SJ_(j), SOJ_(j),SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k),C(═O)J_(j), O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k),N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k);

or q_(e) and q_(f) together are ═C(q_(g))(q_(h));

q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂ alkyl orsubstituted C₁-C₁₂ alkyl.

The synthesis and preparation of the methyleneoxy (4′-CH₂—O-2′) BNAmonomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine anduracil, along with their oligomerization, and nucleic acid recognitionproperties have been described (Koshkin et al., Tetrahedron, 1998, 54,3607-3630). BNAs and preparation thereof are also described in WO98/39352 and WO 99/14226.

Analogs of methyleneoxy (4′-CH₂—O-2′) BNA and 2′-thio-BNAs, have alsobeen prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8,2219-2222). Preparation of locked nucleoside analogs comprisingoligodeoxyribonucleotide duplexes as substrates for nucleic acidpolymerases has also been described (Wengel et al., WO 99/14226).Furthermore, synthesis of 2′-amino-BNA, a novel conformationallyrestricted high-affinity oligonucleotide analog has been described inthe art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Inaddition, 2′-amino- and 2′-methylamino-BNA's have been prepared and thethermal stability of their duplexes with complementary RNA and DNAstrands has been previously reported.

In certain embodiments, bicyclic nucleosides are provided having FormulaVI:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

each q_(i), q_(j), q_(k) and q_(l) is, independently, H, halogen, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxyl,substituted C₁-C₁₂ alkoxyl, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j),NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k), C(═O)J_(j),O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) orN(H)C(═S)NJ_(j)J_(k); and

q_(i) and q_(j) or q_(l) and q_(k) together are ═C(q_(g))(q_(h)),wherein q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂alkyl or substituted C₁-C₁₂ alkyl.

One carbocyclic bicyclic nucleoside having a 4′-(CH₂)₃-2′ bridge and thealkenyl analog bridge 4′-CH═CH—CH₂-2′ have been described (Freier etal., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al.,J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation ofcarbocyclic bicyclic nucleosides along with their oligomerization andbiochemical studies have also been described (Srivastava et al., J. Am.Chem. Soc., 2007, 129(26), 8362-8379).

As used herein, “4′-2′ bicyclic nucleoside” or “4′ to 2′ bicyclicnucleoside” refers to a bicyclic nucleoside comprising a furanose ringcomprising a bridge connecting two carbon atoms of the furanose ringconnects the 2′ carbon atom and the 4′ carbon atom of the sugar ring.

As used herein, “monocyclic nucleosides” refer to nucleosides comprisingmodified sugar moieties that are not bicyclic sugar moieties. In certainembodiments, the sugar moiety, or sugar moiety analogue, of a nucleosidemay be modified or substituted at any position.

As used herein, “2′-modified sugar” means a furanosyl sugar modified atthe 2′ position. In certain embodiments, such modifications includesubstituents selected from: a halide, including, but not limited tosubstituted and unsubstituted alkoxy, substituted and unsubstitutedthioalkyl, substituted and unsubstituted amino alkyl, substituted andunsubstituted alkyl, substituted and unsubstituted allyl, andsubstituted and unsubstituted alkynyl. In certain embodiments, 2′modifications are selected from substituents including, but not limitedto: O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)F,O(CH₂)_(n)ONH₂, OCH₂C(═O)N(H)CH₃, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, wheren and m are from 1 to about 10. Other 2′-substituent groups can also beselected from: C₁-C₁₂ alkyl, substituted alkyl, alkenyl, alkynyl,alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, F,CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving pharmacokinetic properties, or a group for improving thepharmacodynamic properties of an antisense compound, and othersubstituents having similar properties. In certain embodiments, modifiednucleosides comprise a 2′-MOE side chain (Baker et al., J. Biol. Chem.,1997, 272, 11944-12000). Such 2′-MOE substitution have been described ashaving improved binding affinity compared to unmodified nucleosides andto other modified nucleosides, such as 2′-O-methyl, O-propyl, andO-aminopropyl. Oligonucleotides having the 2′-MOE substituent also havebeen shown to be antisense inhibitors of gene expression with promisingfeatures for in vivo use (Martin, Helv. Chim. Acta, 1995, 78, 486-504;Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc.Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides,1997, 16, 917-926).

As used herein, a “modified tetrahydropyran nucleoside” or “modified THPnucleoside” means a nucleoside having a six-membered tetrahydropyran“sugar” substituted in for the pentofuranosyl residue in normalnucleosides (a sugar surrogate). Modified THP nucleosides include, butare not limited to, what is referred to in the art as hexitol nucleicacid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (seeLeumann, Bioorg. Med. Chem., 2002, 10, 841-1954) or fluoro HNA (F-HNA)having a tetrahydropyran ring system as illustrated below:

In certain embodiments, sugar surrogates are selected having FormulaVII:

wherein independently for each of said at least one tetrahydropyrannucleoside analog of Formula VII:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently, an internucleoside linkinggroup linking the tetrahydropyran nucleoside analog to the antisensecompound or one of T_(a) and T_(b) is an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the antisense compoundand the other of T_(a) and T_(b) is H, a hydroxyl protecting group, alinked conjugate group or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl; and each of R₁ and R₂ is selectedfrom hydrogen, hydroxyl, halogen, substituted or unsubstituted alkoxy,NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein Xis O, S or NJ₁ and each J₁, J₂ and J₃ is, independently, H or C₁-C₆alkyl.

In certain embodiments, the modified THP nucleosides of Formula VII areprovided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certainembodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other thanH. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇is methyl. In certain embodiments, THP nucleosides of Formula VII areprovided wherein one of R₁ and R₂ is fluoro. In certain embodiments, R₁is fluoro and R₂ is H; R₁ is methoxy and R₂ is H, and R₁ ismethoxyethoxy and R₂ is H.

In certain embodiments, sugar surrogates comprise rings having more than5 atoms and more than one heteroatom. For example nucleosides comprisingmorpholino sugar moieties and their use in oligomeric compounds has beenreported (see for example: Braasch et al., Biochemistry, 2002, 41,4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and5,034,506). As used here, the term “morpholino” means a sugar surrogatehaving the following formula:

In certain embodiments, morpholinos may be modified, for example byadding or altering various substituent groups from the above morpholinostructure. Such sugar surrogates are referred to herein as “modifiedmorpholinos.”

Combinations of modifications are also provided without limitation, suchas 2′-F-5′-methyl substituted nucleosides (see PCT InternationalApplication WO 2008/101157 published on Aug. 21, 2008 for otherdisclosed 5′, 2′-bis substituted nucleosides) and replacement of theribosyl ring oxygen atom with S and further substitution at the2′-position (see published U.S. Patent Application US2005-0130923,published on Jun. 16, 2005) or alternatively 5′-substitution of abicyclic nucleic acid (see PCT International Application WO 2007/134181,published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside isfurther substituted at the 5′ position with a 5′-methyl or a 5′-vinylgroup). The synthesis and preparation of carbocyclic bicyclicnucleosides along with their oligomerization and biochemical studieshave also been described (see, e.g., Srivastava et al., J. Am. Chem.Soc. 2007, 129(26), 8362-8379).

In certain embodiments, antisense compounds comprise one or moremodified cyclohexenyl nucleosides, which is a nucleoside having asix-membered cyclohexenyl in place of the pentofuranosyl residue innaturally occurring nucleosides. Modified cyclohexenyl nucleosidesinclude, but are not limited to those described in the art (see forexample commonly owned, published PCT Application WO 2010/036696,published on Apr. 10, 2010, Robeyns et al., J. Am. Chem. Soc., 2008,130(6), 1979-1984; Horvath et al., Tetrahedron Letters, 2007, 48,3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30),9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005,24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research, 2005,33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F:Structural Biology and Crystallization Communications, 2005, F61(6),585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111-2123; Gu et al.,Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem.,2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research, 2001,29(24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wanget al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7),785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published PCTapplication, WO 06/047842; and Published PCT Application WO 01/049687;the text of each is incorporated by reference herein, in theirentirety). Certain modified cyclohexenyl nucleosides have Formula X.

wherein independently for each of said at least one cyclohexenylnucleoside analog of Formula X:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the cyclohexenyl nucleoside analog to an antisense compound orone of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to an antisense compound and the otherof T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup, or a 5′- or 3′-terminal group; and

q₁, q₂, q₃, q₄, q₅, q₆, q₇, q₈ and q₉ are each, independently, H, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or other sugarsubstituent group.

As used herein, “2′-modified” or “2′-substituted” refers to a nucleosidecomprising a sugar comprising a substituent at the 2′ position otherthan H or OH. 2′-modified nucleosides, include, but are not limited to,bicyclic nucleosides wherein the bridge connecting two carbon atoms ofthe sugar ring connects the 2′ carbon and another carbon of the sugarring; and nucleosides with non-bridging 2′substituents, such as allyl,amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, —OCF₃, O—(CH₂)₂—O—CH₃,2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)), orO—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is,independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.2′-modified nucleosides may further comprise other modifications, forexample at other positions of the sugar and/or at the nucleobase.

As used herein, “2′-F” refers to a nucleoside comprising a sugarcomprising a fluoro group at the 2′ position of the sugar ring.

As used herein, “2′-OMe” or “2′-OCH₃” or “2′-O-methyl” each refers to anucleoside comprising a sugar comprising an —OCH₃ group at the 2′position of the sugar ring.

As used herein, “MOE” or “2′-MOE” or “2′-OCH₂CH₂OCH₃” or“2′-O-methoxyethyl” each refers to a nucleoside comprising a sugarcomprising a —OCH₂CH₂OCH₃ group at the 2′ position of the sugar ring.

As used herein, “oligonucleotide” refers to a compound comprising aplurality of linked nucleosides. In certain embodiments, one or more ofthe plurality of nucleosides is modified. In certain embodiments, anoligonucleotide comprises one or more ribonucleosides (RNA) and/ordeoxyribonucleosides (DNA).

Many other monocyclo, bicyclo and tricyclo sugar surrogate ring systemsare also known in the art that can be used to modify nucleosides forincorporation into antisense compounds as provided herein (see forexample review article: Leumann, Bioorg. Med. Chem., 2002, 10,841-1954). Such ring systems can undergo various additionalsubstitutions to enhance activity.

Methods for the preparations of modified sugars are well known to thoseskilled in the art. Some representative U.S. patents that teach thepreparation of such modified sugars include without limitation, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,670,633; 5,700,920; 5,792,847 and 6,600,032 and InternationalApplication PCT/US2005/019219, filed Jun. 2, 2005 and published as WO2005/121371 on Dec. 22, 2005, and each of which is herein incorporatedby reference in its entirety.

In nucleotides having modified sugar moieties, the nucleobase moieties(natural, modified or a combination thereof) are maintained forhybridization with an appropriate nucleic acid target.

In certain embodiments, antisense compounds comprise one or morenucleosides having modified sugar moieties. In certain embodiments, themodified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOEmodified nucleosides are arranged in a gapmer motif. In certainembodiments, the modified sugar moiety is a bicyclic nucleoside having a(4′-CH(CH₃)—O-2′) bridging group. In certain embodiments, the(4′-CH(CH₃)—O-2′) modified nucleosides are arranged throughout the wingsof a gapmer motif.

Conjugated Antisense Compounds

Antisense compounds may be covalently linked to one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the resulting antisense oligonucleotides. Typical conjugategroups include cholesterol moieties and lipid moieties. Additionalconjugate groups include carbohydrates, phospholipids, biotin,phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes.

Antisense compounds can also be modified to have one or more stabilizinggroups that are generally attached to one or both termini of antisensecompounds to enhance properties such as, for example, nucleasestability. Included in stabilizing groups are cap structures. Theseterminal modifications protect the antisense compound having terminalnucleic acid from exonuclease degradation, and can help in deliveryand/or localization within a cell. The cap can be present at the5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), or can be presenton both termini. Cap structures are well known in the art and include,for example, inverted deoxy abasic caps. Further 3′ and 5′-stabilizinggroups that can be used to cap one or both ends of an antisense compoundto impart nuclease stability include those disclosed in WO 03/004602published on Jan. 16, 2003.

Cell Culture and Antisense Compounds Treatment

The effects of antisense compounds on the level, activity, or expressionof PKK nucleic acids can be tested in vitro in a variety of cell types.Cell types used for such analyses are available from commercial vendors(e.g., American Type Culture Collection, Manassas, Va.; Zen-Bio, Inc.,Research Triangle Park, N.C.; Clonetics Corporation, Walkersville, Md.)and are cultured according to the vendor's instructions usingcommercially available reagents (e.g., Life Technologies, Carlsbad,Calif.). Illustrative cell types include, but are not limited to,HepaRG™T cells and mouse primary hepatocytes.

In Vitro Testing of Antisense Oligonucleotides

Described herein are methods for treatment of cells with antisenseoligonucleotides, which can be modified appropriately for treatment withother antisense compounds.

Cells may be treated with antisense oligonucleotides when the cellsreach approximately 60-80% confluency in culture.

One reagent commonly used to introduce antisense oligonucleotides intocultured cells includes the cationic lipid transfection reagentLIPOFECTIN (Life Technologies, Carlsbad, Calif.). Antisenseoligonucleotides may be mixed with LIPOFECTIN in OPTI-MEM 1 (LifeTechnologies, Carlsbad, Calif.) to achieve the desired finalconcentration of antisense oligonucleotide and a LIPOFECTINconcentration that may range from 2 to 12 ug/mL per 100 nM antisenseoligonucleotide.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes LIPOFECTAMINE (Life Technologies, Carlsbad,Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE inOPTI-MEM 1 reduced serum medium (Life Technologies, Carlsbad, Calif.) toachieve the desired concentration of antisense oligonucleotide and aLIPOFECTAMINE concentration that may range from 2 to 12 ug/mL per 100 nMantisense oligonucleotide.

Another technique used to introduce antisense oligonucleotides intocultured cells includes electroporation.

Yet another technique used to introduce antisense oligonucleotides intocultured cells includes free uptake of the oligonucleotides by thecells.

Cells are treated with antisense oligonucleotides by routine methods.Cells may be harvested 16-24 hours after antisense oligonucleotidetreatment, at which time RNA or protein levels of target nucleic acidsare measured by methods known in the art and described herein. Ingeneral, when treatments are performed in multiple replicates, the dataare presented as the average of the replicate treatments.

The concentration of antisense oligonucleotide used varies from cellline to cell line. Methods to determine the optimal antisenseoligonucleotide concentration for a particular cell line are well knownin the art. Antisense oligonucleotides are typically used atconcentrations ranging from 1 nM to 300 nM when transfected withLIPOFECTAMINE. Antisense oligonucleotides are used at higherconcentrations ranging from 625 to 20,000 nM when transfected usingelectroporation.

RNA Isolation

RNA analysis can be performed on total cellular RNA or poly(A)+mRNA.Methods of RNA isolation are well known in the art. RNA is preparedusing methods well known in the art, for example, using the TRIZOLReagent (Life Technologies, Carlsbad, Calif.) according to themanufacturer's recommended protocols.

Analysis of Inhibition of Target Levels or Expression

Inhibition of levels or expression of a PKK nucleic acid can be assayedin a variety of ways known in the art. For example, target nucleic acidlevels can be quantitated by, e.g., Northern blot analysis, competitivepolymerase chain reaction (PCR), or quantitative real-time PCR. RNAanalysis can be performed on total cellular RNA or poly(A)+ mRNA.Methods of RNA isolation are well known in the art. Northern blotanalysis is also routine in the art. Quantitative real-time PCR can beconveniently accomplished using the commercially available ABI PRISM7600, 7700, or 7900 Sequence Detection System, available from PE-AppliedBiosystems, Foster City, Calif. and used according to manufacturer'sinstructions.

Quantitative Real-Time PCR Analysis of Target RNA Levels

Quantitation of target RNA levels may be accomplished by quantitativereal-time PCR using the ABI PRISM 7600, 7700, or 7900 Sequence DetectionSystem (PE-Applied Biosystems, Foster City, Calif.) according tomanufacturer's instructions. Methods of quantitative real-time PCR arewell known in the art.

Prior to real-time PCR, the isolated RNA is subjected to a reversetranscriptase (RT) reaction, which produces complementary DNA (cDNA)that is then used as the substrate for the real-time PCR amplification.The RT and real-time PCR reactions are performed sequentially in thesame sample well. RT and real-time PCR reagents may be obtained fromLife Technologies (Carlsbad, Calif.). RT real-time-PCR reactions arecarried out by methods well known to those skilled in the art.

Gene (or RNA) target quantities obtained by real time PCR are normalizedusing either the expression level of a gene whose expression isconstant, such as cyclophilin A, or by quantifying total RNA usingRIBOGREEN (Life Technologies, Inc. Carlsbad, Calif.). Cyclophilin Aexpression is quantified by real time PCR, by being run simultaneouslywith the target, multiplexing, or separately. Total RNA is quantifiedusing RIBOGREEN RNA quantification reagent (Invetrogen, Inc. Eugene,Oreg.). Methods of RNA quantification by RIBOGREEN are taught in Jones,L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR4000 instrument (PE Applied Biosystems) is used to measure RIBOGREENfluorescence.

Probes and primers are designed to hybridize to a PKK nucleic acid.Methods for designing real-time PCR probes and primers are well known inthe art, and may include the use of software such as PRIMER EXPRESSSoftware (Applied Biosystems, Foster City, Calif.).

Analysis of Protein Levels

Antisense inhibition of PKK nucleic acids can be assessed by measuringPKK protein levels. Protein levels of PKK can be evaluated orquantitated in a variety of ways well known in the art, such asimmunoprecipitation, Western blot analysis (immunoblotting),enzyme-linked immunosorbent assay (ELISA), quantitative protein assays,protein activity assays (for example, caspase activity assays),immunohistochemistry, immunocytochemistry or fluorescence-activated cellsorting (FACS). Antibodies directed to a target can be identified andobtained from a variety of sources, such as the MSRS catalog ofantibodies (Aerie Corporation, Birmingham, Mich.), or can be preparedvia conventional monoclonal or polyclonal antibody generation methodswell known in the art.

In Vivo Testing of Antisense Compounds

Antisense compounds, for example, antisense oligonucleotides, are testedin animals to assess their ability to inhibit expression of PKK andproduce phenotypic changes.

In certain embodiments, such phenotypic changes include those associatedwith an inflammatory disease, such as, reduced inflammation,edema/swelling, vascular permeability, and vascular leakage. In certainembodiments, inflammation is measured by measuring the increase ordecrease of edema, temperature, pain, color of tissue, and abdominalfunction in the animal.

In certain embodiments, such phenotypic changes include those associatedwith a thromboembolic disease, such as, prolonged aPTT, prolonged aPTTtime in conjunction with a normal PT, decreased quantity of PlateletFactor 4 (PF-4), and reduced formation of thrombus or increased time forthrombus formation.

Testing may be performed in normal animals, or in experimental diseasemodels. For administration to animals, antisense oligonucleotides areformulated in a pharmaceutically acceptable diluent, such asphosphate-buffered saline. Administration includes parenteral routes ofadministration, such as intraperitoneal, intravenous, and subcutaneous.Calculation of antisense oligonucleotide dosage and dosing frequency iswithin the abilities of those skilled in the art, and depends uponfactors such as route of administration and animal body weight.Following a period of treatment with antisense oligonucleotides, RNA isisolated from liver tissue and changes in PKK nucleic acid expressionare measured.

Certain Indications

In certain embodiments, the invention provides methods of treating anindividual comprising administering one or more pharmaceuticalcompositions as described herein.

In certain embodiments, the individual has an inflammatory disease. Incertain embodiments, the individual is at risk for developing aninflammatory condition, including, but not limited to hereditaryangioedema (HAE), edema, angioedema, swelling, angioedema of the lids,ocular edema, macular edema, and cerebral edema. This includesindividuals with an acquired problem, disease, or disorder that leads toa risk of inflammation, for example, genetic predisposition to aninflammatory condition, environmental factors, and exposure to certainmedications, including, for example, ACE inhibitors and ARBs. In certainembodiments, the individual has been identified as in need ofanti-inflammation therapy. Examples of such individuals include, but arenot limited to those having a mutation in the genetic code forcomplement 1 esterase inhibitor (i.e., C1-INH) or Factor 12. In certainembodiments, an abnormal code can lead to a deficiency in C1-INH (i.e.,type I HAE), an inability of existing C1-INH to function properly (typeII HAE), or hyperfunctional Factor 12 (i.e., type III HAE).

In certain embodiments, the individual has a thromboembolic disease. Incertain embodiments, the individual is at risk for a blood clottingdisorder, including, but not limited to, infarct, thrombosis, embolism,thromboembolism such as deep vein thrombosis, pulmonary embolism,myocardial infarction, and stroke. This includes individuals with anacquired problem, disease, or disorder that leads to a risk ofthrombosis, for example, surgery, cancer, immobility, sepsis,atherosclerosis atrial fibrillation, as well as genetic predisposition,for example, antiphospholipid syndrome and the autosomal dominantcondition, Factor V Leiden. In certain embodiments, the individual hasbeen identified as in need of anticoagulation therapy. Examples of suchindividuals include, but are not limited to, those undergoing majororthopedic surgery (e.g., hip/knee replacement or hip fracture surgery)and patients in need of chronic treatment, such as those suffering fromarterial fibrillation to prevent stroke.

In certain embodiments the invention provides methods forprophylactically reducing PKK expression in an individual. Certainembodiments include treating an individual in need thereof byadministering to an individual a therapeutically effective amount of anantisense compound targeted to a PKK nucleic acid.

In one embodiment, administration of a therapeutically effective amountof an antisense compound targeted to a PKK nucleic acid is accompaniedby monitoring of PKK levels in the serum of an individual, to determinean individual's response to administration of the antisense compound. Anindividual's response to administration of the antisense compound isused by a physician to determine the amount and duration of therapeuticintervention.

In certain embodiments, administration of an antisense compound targetedto a PKK nucleic acid results in reduction of PKK expression by at least15, 20, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, or 99%, or a range defined by any two of these values. Incertain embodiments, pharmaceutical compositions comprising an antisensecompound targeted to PKK are used for the preparation of a medicamentfor treating a patient suffering or susceptible to an inflammatorydisease or thromboembolic disease.

Certain Compositions 1. ISIS 546254

In certain embodiments, ISIS 546254 is characterized as a 5-10-5 MOEgapmer, having a sequence of (from 5′ to 3′) TGCAAGTCTCTTGGCAAACA(incorporated herein as SEQ ID NO: 570), wherein each internucleosidelinkage is a phosphorothioate linkage, each cytosine is a5′-methylcytosine, each of nucleosides 1-5 and 16-20 are2′-O-methoxyethyl modified nucleosides, and each of nucleosides 6-15 are2′-deoxynucleosides.

In certain embodiments, ISIS 546254 is described by the followingchemical notation: Tes Ges mCes Aes Aes Gds Tds mCds Tds mCds Tds TdsGds Gds mCds Aes Aes Aes mCes Ae; wherein,

A=an adenine,

mC=a 5′-methylcytosine

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, ISIS 546254 is described by the followingchemical structure:

Structure 1. ISIS 546254

In certain embodiments, as provided in Example 2 (hereinbelow), ISIS546254 achieved 95% inhibition of human PKK mRNA in cultured HepaRG™cells (density of 20,000 cells per well) when transfected usingelectroporation with 5,000 nM antisense oligonucleotide after atreatment period of 24 hours and measured by quantitative real-time PCRusing human primer probe set RTS3454 and adjusted according to total RNAcontent, as measured by RIBOGREEN®.

In certain embodiments, as provided in Example 5 (see Tables 34 and 41hereinbelow), ISIS 546254 achieved an IC₅₀ of 0.4 μM and 0.3 μM in a 4point dose response curve (0.19 μM, 0.56 μM, 1.67 μM, and 5.0 μM) incultured HepaRG™ cells (density of 20,000 cells per well) whentransfected using electroporation after a treatment period of 16 andmeasured by quantitative real-time PCR using human primer probe setRTS3454 and adjusted according to total RNA content, as measured byRIBOGREEN®.

In certain embodiments, as provided in Example 7 (hereinbelow), ISIS546254 achieved 31%, 55%, 84%, and 83% human PKK mRNA inhibition and 0%,36%, 51%, and 76% human PKK protein inhibition in transgenic miceharboring the human PKK gene sequence when injected subcutaneously twicea week for 3 weeks with 2.5 mg/kg/week, 5.0 mg/kg/week, 10 mg/kg/week or20 mg/kg/week with ISIS 546254.

In certain embodiments, as provided in Example 8 (hereinbelow), ISISI546254 is effective for inhibiting PKK mRNA and protein expression andis tolerable in primates.

2. ISIS 546343

In certain embodiments, ISIS 546343 is characterized as a 5-10-5 MOEgapmer, having a sequence of (from 5′ to 3′) CCCCCTTCTTTATAGCCAGC(incorporated herein as SEQ ID NO: 705), wherein each internucleosidelinkage is a phosphorothioate linkage, each cytosine is a5′-methylcytosine, each of nucleosides 1-5 and 16-20 are2′-O-methoxyethyl modified nucleosides, and each of nucleosides 6-15 are2′-deoxynucleosides.

In certain embodiments, ISIS 546343 is described by the followingchemical notation: mCes mCes mCes mCes mCes Tds Tds mCds Tds Tds Tds AdsTds Ads Gds mCes mCes Aes Ges mCe; wherein,

A=an adenine,

mC=a 5′-methylcytosine;

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate internucleoside linkage.

In certain embodiments, ISIS 546343 is described by the followingchemical structure:

Structure 2. ISIS 546343

In certain embodiments, as provided in Example 2 (see Tables 9 and 10hereinbelow), ISIS 546343 achieved 97% and 91% human PKK mRNA inhibitionin cultured HepaRG™ cells (density of 20,000 cells per well) whentransfected using electroporation with 5,000 nM antisenseoligonucleotide after a treatment period of 24 hours and measured byquantitative real-time PCR using human primer probe set RTS3454 andadjusted according to total RNA content, as measured by RIBOGREEN®.

In certain embodiments, as provided twice in Example 5 (see Tables 34and 41 hereinbelow), ISIS 546343 achieved an IC₅₀ of 0.4 μM in a 4 pointdose response curve (0.19 μM, 0.56 μM, 1.67 μM, and 5.0 μM) in culturedHepaRG™ cells (density of 20,000 cells per well) when transfected usingelectroporation after a treatment period of 16 and measured byquantitative real-time PCR using human primer probe set RTS3454 andadjusted according to total RNA content, as measured by RIBOGREEN®.

In certain embodiments, as provided in Example 7 (hereinbelow), ISIS546343 achieved 46%, 66%, and 86% human PKK mRNA inhibition and 0%, 38%,and 79% human PKK protein inhibition in transgenic mice harboring thehuman PKK gene sequence when injected subcutaneously twice a week for 3weeks with 2.5 mg/kg/week, 5.0 mg/kg/week, 10 mg/kg/week or 20mg/kg/week with ISIS 546343.

In certain embodiments, as provided in Example 8 (hereinbelow), ISISI546343 is effective for inhibiting PKK mRNA and protein expression andis tolerable in primates.

3. ISIS 548048

In certain embodiments, ISIS 548048 is characterized as a modifiedantisense oligonucleotide having the nucleobase sequence (from 5′ to 3′)CGATATCATGATTCCC (incorporated herein as SEQ ID NO: 1666), consisting ofa combination of sixteen 2′-deoxynucleosides, 2′-O-methoxyethyl modifiednucleosides, and cEt modified nucleosides, wherein each of nucleosides1, 2, and 16 are 2′-O-methoxyethyl modified nucleosides, wherein each ofnucleosides 3, 14, and 15 are cEt modified nucleosides, wherein each ofnucleosides 4-13 are 2′-deoxynucleosides, wherein each intemucleosidelinkage is a phosphorothioate intemucleoside linkage, and wherein eachcytosine is a 5′-methylcytosine.

In certain embodiments, ISIS 548048 is described by the followingchemical notation: mCes Ges Aks Tds Ads Tds mCds Ads Tds Gds Ads Tds TdsmCks mCks mCe; wherein,

A=an adenine,

mC=a 5′-methylcytosine;

G=a guanine,

T=a thymine,

e=a 2′-O-methoxyethyl modified nucleoside,

k=a cEt modified nucleoside,

d=a 2′-deoxynucleoside, and

s=a phosphorothioate intemucleoside linkage.

In certain embodiments, ISIS 548048 is described by the followingchemical structure:

Structure 3. ISIS 548048

In certain embodiments, as provided in Example 3 (hereinbelow), ISIS548048 achieved 84% mRNA inhibition in cultured HepaRG™ cells (densityof 20,000 cells per well) when transfected using electroporation with1,000 nM antisense oligonucleotide after a treatment period of 24 hoursand measured by quantitative real-time PCR using human primer probe setRTS3454 and adjusted according to total RNA content, as measured byRIBOGREEN®.

In certain embodiments, as provided in Example 6 (hereinbelow), ISIS548048 achieved an IC₅₀ of 0.1 μM in a 4 point dose response curve (0.11μM, 0.33 μM, 1.00 μM, and 3.00 μM) in cultured HepaRG™ cells (density of20,000 cells per well) when transfected using electroporation after atreatment period of 16 and measured by quantitative real-time PCR usinghuman primer probe set RTS3454 and adjusted according to total RNAcontent, as measured by RIBOGREEN®.

In certain embodiments, as provided in Example 7 (hereinbelow), ISIS548048 achieved 7%, 77%, 72% and 80% human PKK mRNA inhibition and 23%,70%, 89%, and 98% human PKK protein inhibition in transgenic miceharboring the human PKK gene sequence when injected subcutaneously twicea week for 3 weeks with 2.5 mg/kg/week, 5.0 mg/kg/week, 10 mg/kg/week or20 mg/kg/week with ISIS 548048.

In certain embodiments, as provided in Example 8 (hereinbelow), ISISI548048 is effective for inhibiting PKK mRNA and protein expression andis tolerable in primates.

Certain Hotspot Regions 1. Nucleobases 27427-27466 of SEQ ID NO: 10

In certain embodiments, antisense oligonucletoides are designed totarget nucleobases 27427-27466 of SEQ ID NO: 10 (GENBANK Accession No.NT_016354.19 truncated from nucleobases 111693001 to Ser. No.11/730,000). In certain embodiments, nucleobases 27427-27466 of SEQ IDNO: 10 are a hotspot region. In certain embodiments, nucleobases27427-27466 of SEQ ID NO: 10 are targeted by antisense oligonucleotides.In certain embodiments, the antisense oligonucleotides are 15, 16, 17,18, 19, or 20 nucleobases in length. In certain embodiments, theantisense oligonucleotides are gapmers. In certain embodiments, thegapmers are 5-10-5 MOE gapmers, 4-9-4 MOE gapmers, 4-10-4 MOE gapmers,4-10-3 MOE gapmers, 3-10-4 MOE gapmers, or 3-10-3 MOE gapmers. Incertain embodiments, the gapmers are 5-10-5 MOE and cEt gapmers, 4-9-4MOE and cEt gapmers, 4-10-4 MOE and cEt gapmers, 4-10-3 MOE and cEtgapmers, 3-10-4 MOE and cEt gapmers, or 3-10-3 MOE and cEt gapmers. Incertain embodiments, the nucleosides of the antisense olignonucleotidesare linked by phosphorothioate internucleoside linkages.

In certain embodiments, nucleobases 27427-27466 of SEQ ID NO: 10 aretargeted by the following ISIS numbers: 530993, 530994, 530995, 546251,546252, 546253, 546254, 546255, 546256, 547410, 547411, 547978, 547979,547980, and 547981.

In certain embodiments, nucleobases 27427-27466 of SEQ ID NO: 10 aretargeted by the following SEQ ID NOs: 94, 95, 96, 566, 567, 568, 569,570, 571, 572, 573, 1597, 1598, 1599, and 1600.

In certain embodiments, antisense oligonucleotides targeting nucleobases27427-27466 of SEQ ID NO: 10 achieve at least 30%, at least 31%, atleast 32%, at least 33%, at least 34%, at least 35%, at least 36%, atleast 37%, at least 38%, at least 39%, at least 40%, at least 41%, atleast 42%, at least 43%, at least 44%, at least 45%, at least 46%, atleast 47%, at least 48%, at least 49%, at least 50%, at least 51%, atleast 52%, at least 53%, at least 54%, at least 55%, at least 56%, atleast 57%, at least 58%, at least 59%, at least 60%, at least 61%, atleast 62%, at least 63%, at least 64%, at least 65%, at least 66%, atleast 67%, at least 68%, at least 69%, at least 70%, at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 76%, atleast 77%, at least 78%, at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% reduction of PKK and/or proteinlevels in vitro and/or in vivo.

2. Nucleobases 33183-33242 of SEQ ID NO: 10

In certain embodiments, antisense oligonucletoides are designed totarget nucleobases 33183-33242 of SEQ ID NO: 10 (GENBANK Accession No.NT_016354.19 truncated from nucleobases 111693001 to Ser. No.11/730,000). In certain embodiments, nucleobases 33183-33242 of SEQ IDNO: 10 are a hotspot region. In certain embodiments, nucleobases33183-33242 of SEQ ID NO: 10 are targeted by antisense oligonucleotides.In certain embodiments, the antisense oligonucleotides are 15, 16, 17,18, 19, or 20 nucleobases in length. In certain embodiments, theantisense oligonucleotides are gapmers. In certain embodiments, thegapmers are 5-10-5 MOE gapmers, 4-9-4 MOE gapmers, 4-10-4 MOE gapmers,4-10-3 MOE gapmers, 3-10-4 MOE gapmers, or 3-10-3 MOE gapmers. Incertain embodiments, the gapmers are 5-10-5 MOE and cEt gapmers, 4-9-4MOE and cEt gapmers, 4-10-4 MOE and cEt gapmers, 4-10-3 MOE and cEtgapmers, 3-10-4 MOE and cEt gapmers, or 3-10-3 MOE and cEt gapmers. Incertain embodiments, the nucleosides of the antisense olignonucleotidesare linked by phosphorothioate internucleoside linkages.

In certain embodiments, nucleobases 33183-33242 of SEQ ID NO: 10 aretargeted by the following ISIS numbers: 531052, 531053, 531054, 531055,531056, 531057, 531158, 546343, 546345, 547480, 547481, 547482, and547483.

In certain embodiments, nucleobases 33183-33242 of SEQ ID NO: 10 aretargeted by the following SEQ ID NOs: 155, 156, 157, 158, 159, 160, 261,702, 703, 704, 705, 706, and 707.

In certain embodiments, antisense oligonucleotides targeting nucleobases33183-33242 of SEQ ID NO: 10 achieve at least 30%, at least 31%, atleast 32%, at least 33%, at least 34%, at least 35%, at least 36%, atleast 37%, at least 38%, at least 39%, at least 40%, at least 41%, atleast 42%, at least 43%, at least 44%, at least 45%, at least 46%, atleast 47%, at least 48%, at least 49%, at least 50%, at least 51%, atleast 52%, at least 53%, at least 54%, at least 55%, at least 56%, atleast 57%, at least 58%, at least 59%, at least 60%, at least 61%, atleast 62%, at least 63%, at least 64%, at least 65%, at least 66%, atleast 67%, at least 68%, at least 69%, at least 70%, at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 76%, atleast 77%, at least 78%, at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% reduction of PKK mRNA and/orprotein levels in vitro and/or in vivo.

3. Nucleobases 30570-30610 of SEQ ID NO: 10

In certain embodiments, antisense oligonucletoides are designed totarget nucleobases 30570-30610 of SEQ ID NO: 10 (GENBANK Accession No.NT_016354.19 truncated from nucleobases 111693001 to Ser. No.11/730,000). In certain embodiments, nucleobases 30570-30610 of SEQ IDNO: 10 are a hotspot region. In certain embodiments, nucleobases30570-30610 of SEQ ID NO: 10 are targeted by antisense oligonucleotides.In certain embodiments, the antisense oligonucleotides are 15, 16, 17,18, 19, or 20 nucleobases in length. In certain embodiments, theantisense oligonucleotides are gapmers. In certain embodiments, thegapmers are 5-10-5 MOE gapmers, 4-9-4 MOE gapmers, 4-10-4 MOE gapmers,4-10-3 MOE gapmers, 3-10-4 MOE gapmers, or 3-10-3 MOE gapmers. Incertain embodiments, the gapmers are 5-10-5 MOE and cEt gapmers, 4-9-4MOE and cEt gapmers, 4-10-4 MOE and cEt gapmers, 4-10-3 MOE and cEtgapmers, 3-10-4 MOE and cEt gapmers, or 3-10-3 MOE and cEt gapmers. Incertain embodiments, the nucleosides of the antisense olignonucleotidesare linked by phosphorothioate internucleoside linkages.

In certain embodiments, nucleobases 30570-30610 of SEQ ID NO: 10 aretargeted by the following ISIS numbers: 531026, 546309, 546310, 546311,546313, 547453, 547454, 547455, 547456, 547457, 547458, 548046, 548047,548048, 548049, and 548050.

In certain embodiments, nucleobases 30570-30610 of SEQ ID NO: 10 aretargeted by the following SEQ ID NOs: 129, 652, 653, 654, 655, 656, 657,658, 659, 660, 661, 1664, 1665, 1666, 1667, and 1668.

In certain embodiments, antisense oligonucleotides targeting nucleobases30570-30610 of SEQ ID NO: 10 achieve at least 30%, at least 31%, atleast 32%, at least 33%, at least 34%, at least 35%, at least 36%, atleast 37%, at least 38%, at least 39%, at least 40%, at least 41%, atleast 42%, at least 43%, at least 44%, at least 45%, at least 46%, atleast 47%, at least 48%, at least 49%, at least 50%, at least 51%, atleast 52%, at least 53%, at least 54%, at least 55%, at least 56%, atleast 57%, at least 58%, at least 59%, at least 60%, at least 61%, atleast 62%, at least 63%, at least 64%, at least 65%, at least 66%, atleast 67%, at least 68%, at least 69%, at least 70%, at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 76%, atleast 77%, at least 78%, at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% reduction of PKK mRNA and/orprotein levels in vitro and/or in vivo.

4. Nucleobases 27427-27520 of SEQ ID NO: 10

In certain embodiments, antisense oligonucletoides are designed totarget nucleobases 27427-27520 of SEQ ID NO: 10 (GENBANK Accession No.NT_016354.19 truncated from nucleobases 111693001 to Ser. No.11/730,000). In certain embodiments, nucleobases 27427-27520 of SEQ IDNO: 10 are a hotspot region. In certain embodiments, nucleobases27427-27520 of SEQ ID NO: 10 are targeted by antisense oligonucleotides.In certain embodiments, the antisense oligonucleotides are 15, 16, 17,18, 19, or 20 nucleobases in length. In certain embodiments, theantisense oligonucleotides are gapmers. In certain embodiments, thegapmers are 5-10-5 MOE gapmers, 4-9-4 MOE gapmers, 4-10-4 MOE gapmers,4-10-3 MOE gapmers, 3-10-4 MOE gapmers, or 3-10-3 MOE gapmers. Incertain embodiments, the gapmers are 5-10-5 MOE and cEt gapmers, 4-9-4MOE and cEt gapmers, 4-10-4 MOE and cEt gapmers, 4-10-3 MOE and cEtgapmers, 3-10-4 MOE and cEt gapmers, or 3-10-3 MOE and cEt gapmers. Incertain embodiments, the nucleosides of the antisense olignonucleotidesare linked by phosphorothioate internucleoside linkages.

In certain embodiments, nucleobases 27427-27520 of SEQ ID NO: 10 aretargeted by the following ISIS numbers: 530993-530999, 546251-546256,546258-546260, 546263, 546265-546268, 547410-547417, and 547978-547992.

In certain embodiments, nucleobases 27427-27520 of SEQ ID NO: 10 aretargeted by the following SEQ ID NOs: 94-100, 566-587, and 1597-1611.

In certain embodiments, antisense oligonucleotides targeting nucleobases27427-27520 of SEQ ID NO: 10 achieve at least 30%, at least 31%, atleast 32%, at least 33%, at least 34%, at least 35%, at least 36%, atleast 37%, at least 38%, at least 39%, at least 40%, at least 41%, atleast 42%, at least 43%, at least 44%, at least 45%, at least 46%, atleast 47%, at least 48%, at least 49%, at least 50%, at least 51%, atleast 52%, at least 53%, at least 54%, at least 55%, at least 56%, atleast 57%, at least 58%, at least 59%, at least 60%, at least 61%, atleast 62%, at least 63%, at least 64%, at least 65%, at least 66%, atleast 67%, at least 68%, at least 69%, at least 70%, at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 76%, atleast 77%, at least 78%, at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% reduction of PKK and/or proteinlevels in vitro and/or in vivo.

5. Nucleobases 33085-33247 of SEQ ID NO: 10

In certain embodiments, antisense oligonucletoides are designed totarget nucleobases 33085-33247 of SEQ ID NO: 10 (GENBANK Accession No.NT_016354.19 truncated from nucleobases 111693001 to Ser. No.11/730,000). In certain embodiments, nucleobases 33085-33247 of SEQ IDNO: 10 are a hotspot region. In certain embodiments, nucleobases33085-33247 of SEQ ID NO: 10 are targeted by antisense oligonucleotides.In certain embodiments, the antisense oligonucleotides are 15, 16, 17,18, 19, or 20 nucleobases in length. In certain embodiments, theantisense oligonucleotides are gapmers. In certain embodiments, thegapmers are 5-10-5 MOE gapmers, 4-9-4 MOE gapmers, 4-10-4 MOE gapmers,4-10-3 MOE gapmers, 3-10-4 MOE gapmers, or 3-10-3 MOE gapmers. Incertain embodiments, the gapmers are 5-10-5 MOE and cEt gapmers, 4-9-4MOE and cEt gapmers, 4-10-4 MOE and cEt gapmers, 4-10-3 MOE and cEtgapmers, 3-10-4 MOE and cEt gapmers, or 3-10-3 MOE and cEt gapmers. Incertain embodiments, the nucleosides of the antisense olignonucleotidesare linked by phosphorothioate internucleoside linkages.

In certain embodiments, nucleobases 33085-33247 of SEQ ID NO: 10 aretargeted by the following ISIS numbers: 531041-531158, 546336, 546339,546340, 546343, 546345, 547474-547483, 547778, 548077-548082, and548677-548678.

In certain embodiments, nucleobases 33085-33247 of SEQ ID NO: 10 aretargeted by the following SEQ ID NOs: 144-160, 261, 693-707, 1256,1320-1325, 2214, and 2215. In certain embodiments, antisenseoligonucleotides targeting nucleobases 33085-33247 of SEQ ID NO: 10achieve at least 30%, at least 31%, at least 32%, at least 33%, at least34%, at least 35%, at least 36%, at least 37%, at least 38%, at least39%, at least 40%, at least 41%, at least 42%, at least 43%, at least44%, at least 45%, at least 46%, at least 47%, at least 48%, at least49%, at least 50%, at least 51%, at least 52%, at least 53%, at least54%, at least 55%, at least 56%, at least 57%, at least 58%, at least59%, at least 60%, at least 61%, at least 62%, at least 63%, at least64%, at least 65%, at least 66%, at least 67%, at least 68%, at least69%, at least 70%, at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at lest99% reduction of PKK and/or protein levels in vitro and/or in vivo.

6. Nucleobases 30475-30639 of SEQ ID NO: 10

In certain embodiments, antisense oligonucletoides are designed totarget nucleobases 30475-30639 of SEQ ID NO: 10 (GENBANK Accession No.NT_016354.19 truncated from nucleobases 111693001 to Ser. No.11/730,000). In certain embodiments, nucleobases 30475-30639 of SEQ IDNO: 10 are a hotspot region. In certain embodiments, nucleobases30475-30639 of SEQ ID NO: 10 are targeted by antisense oligonucleotides.In certain embodiments, the antisense oligonucleotides are 15, 16, 17,18, 19, or 20 nucleobases in length. In certain embodiments, theantisense oligonucleotides are gapmers. In certain embodiments, thegapmers are 5-10-5 MOE gapmers, 4-9-4 MOE gapmers, 4-10-4 MOE gapmers,4-10-3 MOE gapmers, 3-10-4 MOE gapmers, or 3-10-3 MOE gapmers. Incertain embodiments, the gapmers are 5-10-5 MOE and cEt gapmers, 4-9-4MOE and cEt gapmers, 4-10-4 MOE and cEt gapmers, 4-10-3 MOE and cEtgapmers, 3-10-4 MOE and cEt gapmers, or 3-10-3 MOE and cEt gapmers. Incertain embodiments, the nucleosides of the antisense olignonucleotidesare linked by phosphorothioate internucleoside linkages.

In certain embodiments, nucleobases 30475-30639 of SEQ ID NO: 10 aretargeted by the following ISIS numbers: 531021-531029, 531146, 546297,546299-546304, 546306-546311, 546313, 546316-546319, 547444-547462,548031, 548032, and 548034-548056.

In certain embodiments, nucleobases 30475-30639 of SEQ ID NO: 10 aretargeted by the following SEQ ID NOs: 124-132, 249, 633-669, and1650-1674.

In certain embodiments, antisense oligonucleotides targeting nucleobases30475-30639 of SEQ ID NO: 10 achieve at least 30%, at least 31%, atleast 32%, at least 33%, at least 34%, at least 35%, at least 36%, atleast 37%, at least 38%, at least 39%, at least 40%, at least 41%, atleast 42%, at least 43%, at least 44%, at least 45%, at least 46%, atleast 47%, at least 48%, at least 49%, at least 50%, at least 51%, atleast 52%, at least 53%, at least 54%, at least 55%, at least 56%, atleast 57%, at least 58%, at least 59%, at least 60%, at least 61%, atleast 62%, at least 63%, at least 64%, at least 65%, at least 66%, atleast 67%, at least 68%, at least 69%, at least 70%, at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 76%, atleast 77%, at least 78%, at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% reduction of PKK and/or proteinlevels in vitro and/or in vivo.

7. Nucleobases 27362-27524 of SEQ ID NO: 10

In certain embodiments, antisense oligonucletoides are designed totarget nucleobases 27362-27524 of SEQ ID NO: 10 (GENBANK Accession No.NT_016354.19 truncated from nucleobases 111693001 to Ser. No.11/730,000). In certain embodiments, nucleobases 27362-27524 correspondto exon 9 of PKK (GENBANK Accession No. NT_016354.19 truncated fromnucleobases 111693001 to Ser. No. 11/730,000). In certain embodiments,nucleobases 27362-27524 of SEQ ID NO: 10 are a hotspot region. Incertain embodiments, nucleobases 27362-27524 of SEQ ID NO: 10 aretargeted by antisense oligonucleotides. In certain embodiments, theantisense oligonucleotides are 15, 16, 17, 18, 19, or 20 nucleobases inlength. In certain embodiments, the antisense oligonucleotides aregapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers,4-9-4 MOE gapmers, 4-10-4 MOE gapmers, 4-10-3 MOE gapmers, 3-10-4 MOEgapmers, or 3-10-3 MOE gapmers. In certain embodiments, the gapmers are5-10-5 MOE and cEt gapmers, 4-9-4 MOE and cEt gapmers, 4-10-4 MOE andcEt gapmers, 4-10-3 MOE and cEt gapmers, 3-10-4 MOE and cEt gapmers, or3-10-3 MOE and cEt gapmers. In certain embodiments, the nucleosides ofthe antisense olignonucleotides are linked by phosphorothioateinternucleoside linkages.

In certain embodiments, nucleobases 27361-27524 of SEQ ID NO: 10 aretargeted by the following ISIS numbers: 530985-530999, 546244,546247-546256, 546258-546260, 546263, 546265-546268, 547403-547417,547723, 547968-547970, and 547972-547992.

In certain embodiments, nucleobases 27361-27524 of SEQ ID NO: 10 aretargeted by the following SEQ ID NOs: 86-100, 554-587, 1217, and1588-1611.

In certain embodiments, antisense oligonucleotides targeting nucleobases27362-27524 of SEQ ID NO: 10 achieve at least 30%, at least 31%, atleast 32%, at least 33%, at least 34%, at least 35%, at least 36%, atleast 37%, at least 38%, at least 39%, at least 40%, at least 41%, atleast 42%, at least 43%, at least 44%, at least 45%, at least 46%, atleast 47%, at least 48%, at least 49%, at least 50%, at least 51%, atleast 52%, at least 53%, at least 54%, at least 55%, at least 56%, atleast 57%, at least 58%, at least 59%, at least 60%, at least 61%, atleast 62%, at least 63%, at least 64%, at least 65%, at least 66%, atleast 67%, at least 68%, at least 69%, at least 70%, at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 76%, atleast 77%, at least 78%, at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% reduction of PKK and/or proteinlevels in vitro and/or in vivo.

8. Nucleobases 33101-33240 of SEQ ID NO: 10

In certain embodiments, antisense oligonucletoides are designed totarget nucleobases 33101-33240 of SEQ ID NO: 10 (GENBANK Accession No.NT_016354.19 truncated from nucleobases 111693001 to Ser. No.11/730,000). In certain embodiments, nucleobases 33101-33240 correspondto exon 14 of PKK (GENBANK Accession No. NT_016354.19 truncated fromnucleobases 111693001 to Ser. No. 11/730,000). In certain embodiments,nucleobases 33101-33240 of SEQ ID NO: 10 are a hotspot region. Incertain embodiments, nucleobases 33101-33240 of SEQ ID NO: 10 aretargeted by antisense oligonucleotides. In certain embodiments, theantisense oligonucleotides are 15, 16, 17, 18, 19, or 20 nucleobases inlength. In certain embodiments, the antisense oligonucleotides aregapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers,4-9-4 MOE gapmers, 4-10-4 MOE gapmers, 4-10-3 MOE gapmers, 3-10-4 MOEgapmers, or 3-10-3 MOE gapmers. In certain embodiments, the gapmers are5-10-5 MOE and cEt gapmers, 4-9-4 MOE and cEt gapmers, 4-10-4 MOE andcEt gapmers, 4-10-3 MOE and cEt gapmers, 3-10-4 MOE and cEt gapmers, or3-10-3 MOE and cEt gapmers. In certain embodiments, the nucleosides ofthe antisense olignonucleotides are linked by phosphorothioateinternucleoside linkages.

In certain embodiments, nucleobases 33101-33240 of SEQ ID NO: 10 aretargeted by the following ISIS numbers: 531041-531158, 546336, 546339,546340, 546343, 546345, 547474-547483, 548077-548082, and 548678-548678.

In certain embodiments, nucleobases 33101-33240 of SEQ ID NO: 10 aretargeted by the following SEQ ID NOs: 144-160, 261, 693-707, 1320-1325,and 2215.

In certain embodiments, antisense oligonucleotides targeting nucleobases33101-33240 of SEQ ID NO: 10 achieve at least 30%, at least 31%, atleast 32%, at least 33%, at least 34%, at least 35%, at least 36%, atleast 37%, at least 38%, at least 39%, at least 40%, at least 41%, atleast 42%, at least 43%, at least 44%, at least 45%, at least 46%, atleast 47%, at least 48%, at least 49%, at least 50%, at least 51%, atleast 52%, at least 53%, at least 54%, at least 55%, at least 56%, atleast 57%, at least 58%, at least 59%, at least 60%, at least 61%, atleast 62%, at least 63%, at least 64%, at least 65%, at least 66%, atleast 67%, at least 68%, at least 69%, at least 70%, at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 76%, atleast 77%, at least 78%, at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% reduction of PKK and/or proteinlevels in vitro and/or in vivo.

9. Nucleobases 30463-30638 of SEQ ID NO: 10

In certain embodiments, antisense oligonucletoides are designed totarget nucleobases 30463-30638 of SEQ ID NO: 10 (GENBANK Accession No.NT_016354.19 truncated from nucleobases 111693001 to Ser. No.11/730,000). In certain embodiments, nucleobases 30463-30638 correspondto exon 12 of PKK (GENBANK Accession No. NT_016354.19 truncated fromnucleobases 111693001 to Ser. No. 11/730,000). In certain embodiments,nucleobases 30463-30638 of SEQ ID NO: 10 are a hotspot region. Incertain embodiments, nucleobases 30463-30638 of SEQ ID NO: 10 aretargeted by antisense oligonucleotides. In certain embodiments, theantisense oligonucleotides are 15, 16, 17, 18, 19, or 20 nucleobases inlength. In certain embodiments, the antisense oligonucleotides aregapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers,4-9-4 MOE gapmers, 4-10-4 MOE gapmers, 4-10-3 MOE gapmers, 3-10-4 MOEgapmers, or 3-10-3 MOE gapmers. In certain embodiments, the gapmers are5-10-5 MOE and cEt gapmers, 4-9-4 MOE and cEt gapmers, 4-10-4 MOE andcEt gapmers, 4-10-3 MOE and cEt gapmers, 3-10-4 MOE and cEt gapmers, or3-10-3 MOE and cEt gapmers. In certain embodiments, the nucleosides ofthe antisense olignonucleotides are linked by phosphorothioateinternucleoside linkages.

In certain embodiments, nucleobases 30463-30638 of SEQ ID NO: 10 aretargeted by the following ISIS numbers: 531021-531029, 531146, 546297,546299-546304, 546306-546311, 546313, 546316-546319, 547444-547462,548031, 548032, and 548034-548056.

In certain embodiments, nucleobases 30463-30638 of SEQ ID NO: 10 aretargeted by the following SEQ ID NOs: 124-132, 249, 633-669, and1650-1674.

In certain embodiments, antisense oligonucleotides targeting nucleobases30463-30638 of SEQ ID NO: 10 achieve at least 30%, at least 31%, atleast 32%, at least 33%, at least 34%, at least 35%, at least 36%, atleast 37%, at least 38%, at least 39%, at least 40%, at least 41%, atleast 42%, at least 43%, at least 44%, at least 45%, at least 46%, atleast 47%, at least 48%, at least 49%, at least 50%, at least 51%, atleast 52%, at least 53%, at least 54%, at least 55%, at least 56%, atleast 57%, at least 58%, at least 59%, at least 60%, at least 61%, atleast 62%, at least 63%, at least 64%, at least 65%, at least 66%, atleast 67%, at least 68%, at least 69%, at least 70%, at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 76%, atleast 77%, at least 78%, at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% reduction of PKK and/or proteinlevels in vitro and/or in vivo.

EXAMPLES Non-Limiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the referencesrecited in the present application is incorporated herein by referencein its entirety.

Example 1: Antisense Inhibition of Human PKK in HepaRG™^(T) Cells byAntisense Oligonucleotides with 2′-MOE Sugar Modifications

Antisense oligonucleotides were designed targeting a PKK nucleic acidand were tested for their effects on PKK mRNA in vitro. HepaRG™ cells,which are terminally differentiated hepatic cells derived from a humanhepatic progenitor cell line and retain many characteristics of primaryhuman hepatocytes (Lubberstedt M. et al., J. Pharmacol. Toxicol. Methods2011 63: 59-68), were used in the screen.

The chimeric antisense oligonucleotides in the tables below weredesigned as 5-10-5 MOE gapmers. The gapmers are 20 nucleosides inlength, wherein the central gap segment comprises often2′-deoxynucleosides and is flanked by wing segments on the 5′ directionand the 3′ direction comprising five nucleosides each. Each nucleosidein the 5′ wing segment and each nucleoside in the 3′ wing segment has a2′-O-methoxyethyl modification. The internucleoside linkages throughouteach gapmer are phosphorothioate linkages. All cytosine residuesthroughout each gapmer are 5-methylcytosines. “Start site” indicates the5′-most nucleoside to which the gapmer is targeted in the human genesequence. “Stop site” indicates the 3′-most nucleoside to which thegapmer is targeted in the human gene sequence. Each gapmer listed in thetables below is targeted to either the human PKK mRNA, designated hereinas SEQ ID NO: 1 (GENBANK Accession No. NM_000892.3) or the human PKKgenomic sequence, designated herein as SEQ ID NO: 10 (GENBANK AccessionNo. NT_016354.19 truncated from nucleotides 111693001 to Ser. No.11/730,000). ‘n/a’ indicates that the antisense oligonucleotide does nottarget that particular gene sequence.

Cultured HepaRG™ cells at a density of 20,000 cells per well weretransfected using electroporation with 3,000 nM antisenseoligonucleotide. After a treatment period of approximately 24 hours, RNAwas isolated from the cells and PKK mRNA levels were measured byquantitative real-time PCR. Human primer probe set RTS3454 (forwardsequence CCAAAAAAGGTGCACCAGTAACA, designated herein as SEQ ID NO: 20;reverse sequence CCTCCGGGACTGTACTTTAATAGG, designated herein as SEQ IDNO: 21; probe sequence CACGCAAACATTTCACAAGGCAGAGTACC, designated hereinas SEQ ID NO: 22) was used to measure mRNA levels. PKK mRNA levels wereadjusted according to total RNA content, as measured by RIBOGREEN®. Theantisense oligonucleotides were tested in a series of experiments thathad similar culture conditions. The results for each experiment arepresented in separate tables shown below. Results are presented aspercent inhibition of PKK, relative to untreated control cells.

TABLE 1 SEQ SEQ ID ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 10 NO: 10 SEQ StartStop % Start Stop ID ISIS NO Site Site Sequence inhibition Site Site NO530929 1 20 AACGGTCTTCAAGCTGTTCT 59 3393 3412 30 530930 6 25AAATGAACGGTCTTCAAGCT 17 3398 3417 31 530931 11 30 CTTAAAAATGAACGGTCTTC29 3403 3422 32 530932 16 35 TGTCACTTAAAAATGAACGG 52 3408 3427 33 53093331 50 TGGAGGTGAGTCTCTTGTCA 76 3423 3442 34 530934 36 55CTTCTTGGAGGTGAGTCTCT 54 3428 3447 35 530935 68 87 GCTTGAATAAAATCATTCTG 0n/a n/a 36 530936 73 92 TGCTTGCTTGAATAAAATCA 27 4072 4091 37 530937 7897 TAAGTTGCTTGCTTGAATAA 0 4077 4096 38 530938 88 107GGAAATGAAATAAGTTGCTT 11 4087 4106 39 530939 93 112 AACAAGGAAATGAAATAAGT0 4092 4111 40 530940 98 117 TAGCAAACAAGGAAATGAAA 7 4097 4116 41 530941103 122 AACTGTAGCAAACAAGGAAA 22 4102 4121 42 530942 108 127CAGGAAACTGTAGCAAACAA 22 4107 4126 43 530943 113 132 ATCCACAGGAAACTGTAGCA56 n/a n/a 44 530944 118 137 CAGACATCCACAGGAAACTG 0 n/a n/a 45 530945157 176 ATCCCCACCTCTGAAGAAGG 0 8029 8048 46 530946 160 179TACATCCCCACCTCTGAAGA 0 8032 8051 47 530947 165 184 GAAGCTACATCCCCACCTCT27 8037 8056 48 530948 170 189 ACATGGAAGCTACATCCCCA 35 8042 8061 49530949 175 194 GGTGTACATGGAAGCTACAT 31 8047 8066 50 530950 221 240ACCTTGGGTGGAATGTGCAC 47 8093 8112 51 530951 226 245 CAAACACCTTGGGTGGAATG49 8098 8117 52 530952 234 253 CTGAATAGCAAACACCTTGG 38 8106 8125 53530953 239 258 GAAAACTGAATAGCAAACAC 7 8111 8130 54 530954 244 263TGGAAGAAAACTGAATAGCA 47 8116 8135 55 530955 278 297 CAAACCTTTTCTCCATGTCA55 n/a n/a 56 530956 300 319 ACACTATCTTTCAAGAAGCA 57 9834 9853 57 530957386 405 GGCAAGCACTTATTTGATGA 56 n/a n/a 58 530958 432 451TTAAAATTGACTCCTCTCAT 60 12688 12707 59 530959 456 475TCAACACTGCTAACCTTAGA 60 12712 12731 60 530960 461 480ATTCTTCAACACTGCTAACC 58 12717 12736 61 530961 466 485TTGGCATTCTTCAACACTGC 88 12722 12741 62 530962 472 491CCTTTTTTGGCATTCTTCAA 64 12728 12747 63 530963 479 498TGGTGCACCTTTTTTGGCAT 78 12735 12754 64 530964 628 647CTTCAGTGAGAATCCAGATT 44 14199 14218 65 530965 637 656GGCACAGGGCTTCAGTGAGA 73 14208 14227 66 530966 649 668AATTTCTGAAAGGGCACAGG 58 14220 14239 67 530967 654 673CAACCAATTTCTGAAAGGGC 69 n/a n/a 68 530968 680 699 CAAGATGCTGGAAGATGTTC18 26128 26147 69 530969 846 865 GTGCCACTTTCAGATGTTTT 0 27110 27129 70530970 851 870 TTGGTGTGCCACTTTCAGAT 74 27115 27134 71 530971 856 875GGAACTTGGTGTGCCACTTT 85 27120 27139 72 530972 861 880GTAGAGGAACTTGGTGTGCC 42 27125 27144 73 530973 866 885GAGGAGTAGAGGAACTTGGT 52 27130 27149 74 530974 871 890TTCTTGAGGAGTAGAGGAAC 18 27135 27154 75 530975 876 895GTGTTTTCTTGAGGAGTAGA 41 27140 27159 76 530976 881 900ATATGGTGTTTTCTTGAGGA 26 27145 27164 77 530977 886 905TCCAGATATGGTGTTTTCTT 55 27150 27169 78 530978 891 910CTATATCCAGATATGGTGTT 0 27155 27174 79 530979 901 920GGTTAAAAGGCTATATCCAG 35 27165 27184 80 530980 906 925TTGCAGGTTAAAAGGCTATA 29 27170 27189 81 530981 911 930TTCTTTTGCAGGTTAAAAGG 0 27175 27194 82 530982 916 935TAAAGTTCTTTTGCAGGTTA 0 27180 27199 83 530983 931 950ATGGCAGGGTTCAGGTAAAG 9 n/a n/a 84 530984 936 955 TTAGAATGGCAGGGTTCAGG 25n/a n/a 85 530985 941 960 AAATTTTAGAATGGCAGGGT 32 27363 27382 86 530986946 965 CGGGTAAATTTTAGAATGGC 62 27368 27387 87 530987 951 970ACTCCCGGGTAAATTTTAGA 0 27373 27392 88 530988 961 980TCCAAAGTCAACTCCCGGGT 76 27383 27402 89 530989 966 985TCTCCTCCAAAGTCAACTCC 28 27388 27407 90 530990 971 990ATTCTTCTCCTCCAAAGTCA 32 27393 27412 91 530991 976 995ATTCAATTCTTCTCCTCCAA 43 27398 27417 92 530992 981 1000GTCACATTCAATTCTTCTCC 70 27403 27422 93 530993 1005 1024CAAACATTCACTCCTTTAAC 30 27427 27446 94 530994 1010 1029CTTGGCAAACATTCACTCCT 50 27432 27451 95 530995 1015 1034AGTCTCTTGGCAAACATTCA 49 27437 27456 96 530996 1038 1057TGACAGCGAATCATCTTTGT 51 27460 27479 97 530997 1043 1062AAAACTGACAGCGAATCATC 39 27465 27484 98 530998 1048 1067AGTGAAAAACTGACAGCGAA 0 27470 27489 99 530999 1071 1090CAGTCTTCTGGGAGTAAAGA 31 27493 27512 100 531000 1098 1117AAGAAACACTTACACTTCTC 1 n/a n/a 101 531001 1108 1127 AGATAATCTTAAGAAACACT44 27629 27648 102 531002 1155 1174 GAGCTCCCTTGTGTCCCATA 85 27676 27695103 531003 1160 1179 AACCAGAGCTCCCTTGTGTC 49 27681 27700 104 531004 11651184 AGAGTAACCAGAGCTCCCTT 76 27686 27705 105 531005 1170 1189CTCAAAGAGTAACCAGAGCT 76 27691 27710 106 531006 1216 1235GCTTGTTTTTGTTGTGCAGA 49 27892 27911 107

TABLE 2 SEQ SEQ ID ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 10 NO: 10 SEQ ISISStart Stop % Start Stop ID NO Site Site Sequence inhibition Site Site NO482586 1608 1627 ACCCAACAGTTGGTATAAAT 0 31914 31933 108 486847 1563 1582AGGCATATTGGTTTTTGGAA 78 31869 31888 109 531007 46 65AACACAATTGCTTCTTGGAG 51 3438 3457 110 531008 675 694TGCTGGAAGATGTTCATGTG 51 26123 26142 111 531009 1239 1258TTTGTTCCTCCAACAATGCG 65 27915 27934 112 531010 1244 1263AAGAGTTTGTTCCTCCAACA 52 27920 27939 113 531011 1249 1268CCAAGAAGAGTTTGTTCCTC 0 27925 27944 114 531012 1254 1273TCTCCCCAAGAAGAGTTTGT 48 27930 27949 115 531013 1264 1283CCAGGGCCACTCTCCCCAAG 56 27940 27959 116 531014 1287 1306AGCTTCACCTGCAGGCTCAC 0 27963 27982 117 531015 1324 1343TATGAGTGACCCTCCACACA 52 28000 28019 118 531016 1329 1348TGTCCTATGAGTGACCCTCC 39 28005 28024 119 531017 1334 1353ACTGGTGTCCTATGAGTGAC 31 28010 28029 120 531018 1339 1358GACCCACTGGTGTCCTATGA 54 28015 28034 121 531019 1344 1363GTGAGGACCCACTGGTGTCC 28 28020 28039 122 531020 1369 1388AAGCCCATCAAAGCAGTGGG 0 n/a n/a 123 531021 1420 1439 GTCTGACAGATTTAAAATGC50 30498 30517 124 531022 1425 1444 GTAATGTCTGACAGATTTAA 74 30503 30522125 531023 1430 1449 CTTTTGTAATGTCTGACAGA 71 30508 30527 126 531024 14521471 TTTATTTGTGAGAAAGGTGT 69 30530 30549 127 531025 1457 1476TCTCTTTTATTTGTGAGAAA 34 30535 30554 128 531026 1501 1520ATCATGATTCCCTTCTGAGA 73 30579 30598 129 531027 1530 1549AAAGGAGCCTGGAGTTTTAT 0 30608 30627 130 531028 1535 1554AATTCAAAGGAGCCTGGAGT 56 30613 30632 131 531029 1540 1559AGTGTAATTCAAAGGAGCCT 59 30618 30637 132 531030 1545 1564AATTCAGTGTAATTCAAAGG 24 n/a n/a 133 531031 1550 1569TTTGGAATTCAGTGTAATTC 59 n/a n/a 134 531032 1555 1574TGGTTTTTGGAATTCAGTGT 67 n/a n/a 135 531033 1557 1576ATTGGTTTTTGGAATTCAGT 53 n/a n/a 136 531034 1560 1579CATATTGGTTTTTGGAATTC 36 31866 31885 137 531035 1565 1584GTAGGCATATTGGTTTTTGG 46 31871 31890 138 531036 1581 1600GTGTCACCTTTGGAAGGTAG 71 31887 31906 139 531037 1604 1623AACAGTTGGTATAAATTGTG 35 31910 31929 140 531038 1605 1624CAACAGTTGGTATAAATTGT 22 31911 31930 141 531039 1609 1628TACCCAACAGTTGGTATAAA 36 31915 31934 142 531040 1632 1651TCCTTCGAGAAGCCCCATCC 27 31938 31957 143 531041 1677 1696AAAGGAATATTTACCTTTTG 68 33121 33140 144 531042 1682 1701TTACCAAAGGAATATTTACC 11 33126 33145 145 531043 1687 1706ATTTGTTACCAAAGGAATAT 27 33131 33150 146 531044 1697 1716GGCATTCTTCATTTGTTACC 68 33141 33160 147 531045 1702 1721TTTCTGGCATTCTTCATTTG 37 33146 33165 148 531046 1709 1728GATATCTTTTCTGGCATTCT 54 33153 33172 149 531047 1714 1733ATCTTGATATCTTTTCTGGC 68 33158 33177 150 531048 1719 1738TTATAATCTTGATATCTTTT 42 33163 33182 151 531049 1724 1743TTATTTTATAATCTTGATAT 2 33168 33187 152 531050 1729 1748TTGGGTTATTTTATAATCTT 18 33173 33192 153 531051 1734 1753ATCCGTTGGGTTATTTTATA 51 33178 33197 154 531052 1739 1758AGACCATCCGTTGGGTTATT 60 33183 33202 155 531053 1744 1763AGCACAGACCATCCGTTGGG 49 33188 33207 156 531054 1754 1773CTTTATAGCCAGCACAGACC 48 33198 33217 157 531055 1759 1778CCCTTCTTTATAGCCAGCAC 68 33203 33222 158 531056 1764 1783TTTCCCCCTTCTTTATAGCC 45 33208 33227 159 531057 1769 1788CATCTTTTCCCCCTTCTTTA 48 33213 33232 160 531058 1779 1798CCCTTACAAGCATCTTTTCC 60 n/a n/a 161 531059 n/a n/a ACATTCCATTGTGTTTGCAA55 33919 33938 162 531060 n/a n/a TGGTGATGCCCACCAAACGC 35 33940 33959163 531061 1872 1891 TGCTCCCTGCGGGCACAGCC 52 33971 33990 164 531062 18771896 CAGGTTGCTCCCTGCGGGCA 39 33976 33995 165 531063 1882 1901GACACCAGGTTGCTCCCTGC 51 33981 34000 166 531064 1887 1906GTGTAGACACCAGGTTGCTC 56 33986 34005 167 531065 1892 1911CTTTGGTGTAGACACCAGGT 57 33991 34010 168 531066 1897 1916AGCGACTTTGGTGTAGACAC 67 33996 34015 169 531067 1902 1921TACTCAGCGACTTTGGTGTA 31 34001 34020 170 531068 1907 1926CCATGTACTCAGCGACTTTG 59 34006 34025 171 531069 1912 1931CCAGTCCATGTACTCAGCGA 56 34011 34030 172 531070 1930 1949CTGTGTTTTCTCTAAAATCC 68 34029 34048 173 531071 1935 1954CTGCTCTGTGTTTTCTCTAA 73 34034 34053 174 531072 2026 2045GCTCAGAATTTGACTTGAAC 64 34125 34144 175 531073 2031 2050CCCAGGCTCAGAATTTGACT 51 34130 34149 176 531074 2049 2068CTTTGCAGATGAGGACCCCC 67 34148 34167 177 531075 2054 2073CCATGCTTTGCAGATGAGGA 64 34153 34172 178 531076 2059 2078ACTCTCCATGCTTTGCAGAT 68 34158 34177 179 531077 2064 2083ATGCCACTCTCCATGCTTTG 51 34163 34182 180 531078 2111 2130AGCAGCTCTGAGTGCACTGT 77 34210 34229 181 531079 2116 2135TCCTCAGCAGCTCTGAGTGC 58 34215 34234 182 531080 2121 2140CATTGTCCTCAGCAGCTCTG 55 34220 34239 183 531081 n/a n/aTGGTTTTTGGAATTCTGAAA 14 31861 31880 184 531082 n/a n/aATATTGGTTTTTGGAATTCT 31 31865 31884 185

TABLE 3 SEQ SEQ ID ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 10 NO: 10 SEQ ISISStart Stop % Start Stop ID NO Site Site Sequence inhibition Site Site NO531083 n/a n/a TGTACTAGTTTCCTATAACT 60 14738 14757 186 14809 14828 1488014899 14939 14958 15071 15090 15214 15233 15286 15305 15345 15364 1547715496 15549 15568 15607 15626 15679 15698 15809 15828 15881 15900 1593915958 531084 n/a n/a ATAGGGACACAACCAAGGAA 25 16296 16315 187 531085 n/an/a AGGCACAGAGCCAGCACCCA 9 16495 16514 188 531086 n/a n/aCCTGCCTCCTGGCAGCCTTC 48 16696 16715 189 531087 n/a n/aCCAGGTGTGGACAGCAGCTG 52 16821 16840 190 531088 n/a n/aGGTTTTGTTTGTAAAATTAG 27 17159 17178 191 531089 n/a n/aAAAACACCATTAAATCCATT 45 17306 17325 192 531090 n/a n/aACAGAAACCATGATGTTGCT 59 17644 17663 193 531091 n/a n/aTCAGCCCAATGTCCTAACCT 35 17793 17812 194 531092 n/a n/aCCTTCACTGACTCTCTTTTC 24 17922 17941 195 531093 n/a n/aTTCTCCTGGCTCAGAAGCTC 60 18053 18072 196 23315 23334 531094 n/a n/aGAATGTCAGGCCTCTGGGCC 48 18181 18200 197 531095 n/a n/aCTAACAACCCCACAATATCA 20 18390 18409 198 531096 n/a n/aCCCAATTCTTAGTCCTTTAA 45 18523 18542 199 531097 n/a n/aACCAAGCTCAGCCTCCAACT 41 18648 18667 200 531098 n/a n/aTTATTAGTCAAATCACCCAA 19 18773 18792 201 531099 n/a n/aTGGATGGGTAGAGGCCTTTC 64 18898 18917 202 531100 n/a n/aCCCCCTCCCTTCCCTACACA 0 19023 19042 203 531101 n/a n/aATGTAAGTTACAAGCCACTA 37 19153 19172 204 531102 n/a n/aTGCCTCTTTAATAAAAACTC 42 19484 19503 205 531103 n/a n/aACTCATTGCCTTAACTCAGG 40 19636 19655 206 531104 n/a n/aACTTGACCTTACTGTTTTAG 20 19886 19905 207 531105 n/a n/aCTCCTCCCCAGGCTGCTCCT 16 22092 22111 208 531106 n/a n/aAAGATCTAGATAATTCTTGT 31 22332 22351 209 531107 n/a n/aTCAACTCACACCTGACCTAA 30 22457 22476 210 531108 n/a n/aTGAACCCAAAACTCTGGCAC 50 22771 22790 211 531109 n/a n/aAGCCCAAGGAACATCTCACC 52 22959 22978 212 531110 n/a n/aGCCTGTTTGGTGGTCTCTTC 86 23110 23129 213 531111 n/a n/aCTTCTCCTGGCTCAGAAGCT 68 18054 18073 214 23316 23335 531112 n/a n/aATGTATGATTCTAAGAACTT 14 23479 23498 215 531113 n/a n/aAACAGACACATTATTTATAT 0 23604 23623 216 531114 n/a n/aAGAGTCAAGTCCACAGACAT 40 24246 24265 217 531115 n/a n/aTCCTAAATAGGAACAAAGTA 0 24372 24391 218 531116 n/a n/aTTGTTAAGGTTGTAGAGAGA 23 24688 24707 219 531117 n/a n/aACCCAATTATTTTTAATGGC 62 24876 24895 220 531118 n/a n/aGCCTAAATGTAAGAGCTAAA 26 25157 25176 221 531119 n/a n/aTAAACTCTTACATTTATAGA 0 25293 25312 222 531120 n/a n/aAAATAAAAGCACTCAGACTG 0 25418 25437 223 531121 n/a n/aTTGGTCTACAGATTCAATGC 72 25550 25569 224 531122 n/a n/aTAACAAAAATGCCTTGTGCC 33 25710 25729 225 531123 n/a n/aTCCCAGCTCCAGTCACCACC 74 25866 25885 226 531124 n/a n/aGTACTAAACATCCTAAGTGA 2 25992 26011 227 531125 n/a n/aACTCGCCTTTGTGACTCGAT 23 26264 26283 228 531126 n/a n/aTTTTGAATCTTCATTCAAAG 0 26551 26570 229 531127 n/a n/aCAGAGCCTTGATCAGAATAA 12 26676 26695 230 531128 n/a n/aAAGTTCCACCTTCTAACTGG 18 26831 26850 231 531129 n/a n/aAGCAGCTCACACCCAAAAAG 0 27005 27024 232 531130 n/a n/aTTCTGTGTCAATTATAAACA 0 27344 27363 233 531131 n/a n/aTAGAAAGAGTAAGCCTTCAC 0 27587 27606 234 531132 n/a n/aAGTGAGGTTACTCACCAGAG 0 27732 27751 235 531133 n/a n/aTTTTGTTGTGCAGACTGAAA 19 27886 27905 236 531134 n/a n/aTTACCCATCAAAGCAGTGGG 6 28045 28064 237 531135 n/a n/aAATGTTGTGAATACCATCCC 16 28174 28193 238 531136 n/a n/aTAACATTTCTATGGGCCTGA 6 28670 28689 239 531137 n/a n/aTGTCTACTATTTGACCAATA 19 28795 28814 240 531138 n/a n/aTTTAAATGTGTCACTTAATC 0 28987 29006 241 531139 n/a n/aTCACTAAAACAAAAATACTT 0 29156 29175 242 531140 n/a n/aTCTTCCAGGCCAACCACCTT 22 29321 29340 243 531141 n/a n/aTGCAAGGCATGTGTGCACAA 47 29532 29551 244 531142 n/a n/aTGTTTAAAATATCTCTATAC 8 30008 30027 245 531143 n/a n/aCATGGAAAAATTAAGCTCAT 0 30133 30152 246 531144 n/a n/aTGAAGATTCTATTTAACAAA 0 30266 30285 247 531145 n/a n/aGCCTAGGAGAGAAAAATAAA 0 30445 30464 248 531146 n/a n/aCCAGTGTAATTCAAAGGAGC 40 30620 30639 249 531147 n/a n/aCCATTATTTCCATCACCTGC 18 30871 30890 250 531148 n/a n/aTACCCAAATTATACCTGGAA 8 31015 31034 251 531149 n/a n/aAGAGGTAAAGCAACTTGCCC 45 31429 31448 252 531150 n/a n/aTCCTTAATAGTCATAGCAGG 48 31558 31577 253 531151 n/a n/aTCACCACCATTTTTCACATG 44 31683 31702 254 531152 n/a n/aGTTATGGATATAGACTTTAA 0 31808 31827 255 531153 n/a n/aCTAGAAGCAATATTTAAAGC 0 31974 31993 256 531154 n/a n/aATGAAGTAAGATGCTTAAAA 16 32162 32181 257 531155 n/a n/aCTTCTTGTCTCAGATTACCA 79 32464 32483 258 531156 n/a n/aTCTGAAAAGCCCTCCGAGCT 0 32589 32608 259 531157 n/a n/aAAGTGAATCAGAGCAGTGTA 46 32961 32980 260 531158 n/a n/aACCTTACAAGCATCTTTTCC 41 33223 33242 261 531159 n/a n/aATTTGTTAAAAGTTGCTTAT 0 33368 33387 262 531160 n/a n/aTGATATCATCATCCCAATGA 13 33510 33529 263

TABLE 4 SEQ SEQ ID ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 10 NO: 10 SEQ ISISStart Stop % Start Stop ID NO Site Site Sequence inhibition Site Site NO531083 n/a n/a TGTACTAGTTTCCTATAACT 68 14738 14757 264 14809 14828 1488014899 14939 14958 15071 15090 15214 15233 15286 15305 15345 15364 1547715496 15549 15568 15607 15626 15679 15698 15809 15828 15881 15900 1593915958 531161 n/a n/a CAGACACCTTCTTCACAAGG 40 898 917 264 531162 n/a n/aAATTTCCCAGATGTATTAGT 43 1054 1073 265 531163 n/a n/aTCAGCAGAAATCATGTAGGC 60 1181 1200 266 531164 n/a n/aTTAAATATAAAGAGATCCTC 38 1609 1628 267 531165 n/a n/aGTAATAAAAGGAATGATAAA 0 1825 1844 268 531166 n/a n/a AGACAGTAAACAAAATCAGG12 2046 2065 269 531167 n/a n/a CAAGAAACCACCAAAGGAAG 37 2176 2195 270531168 n/a n/a ACCCCAACAGACAGCCCACC 55 2314 2333 271 531169 n/a n/aTGGGCTCACCCCAGTGGACC 54 2580 2599 272 531170 n/a n/aGCCTGGCCCCCAAGACTCTA 54 2743 2762 273 531171 n/a n/aAGGCCTGCCACAGGCCAGAC 40 2873 2892 274 531172 n/a n/aTTCAAGCCTGGGCAGCACAG 71 3004 3023 275 531173 n/a n/aAAAATAACTTCACTAGAGCT 22 3131 3150 276 531174 n/a n/aTGTTAAGTATATTAACTATT 10 3256 3275 277 531175 n/a n/aTACTCAGGAAATTAGAATAT 25 3550 3569 278 531176 n/a n/aTTATGAAACCTCTTGATTTG 0 3753 3772 279 531177 n/a n/a TTCTTGTAAATGTCTGAATT61 3971 3990 280 531178 n/a n/a ACCACAGGAAACTGTAGCAA 72 4111 4130 281531179 n/a n/a GATTGGACCCAGACACTATA 57 4506 4525 282 531180 n/a n/aCCTCTTAAGTCACCATAGAC 45 4785 4804 283 531181 n/a n/aGGTTGAGGGACAGACACAGG 36 4940 4959 284 531182 n/a n/aATAATCATGATTTATTTTGC 34 5099 5118 285 531183 n/a n/aCATAAGAATGTGCACACAAA 39 5382 5401 286 531184 n/a n/aACTCTTATTAGCTGGTAGAA 74 5538 5557 287 531185 n/a n/aGGACCAAAACTGAGAGGCAG 63 5663 5682 288 531186 n/a n/aCCATTACTCTCAAGCTCCAC 75 5890 5909 289 531187 n/a n/aATCTATTGGTTCAGGAGCCA 72 6015 6034 290 531188 n/a n/aGTTAAAACAACTAGAAGCCA 67 6146 6165 291 531189 n/a n/aAGGTGTTCTTGCTTATCCTC 63 6484 6503 292 531190 n/a n/aGCAGTCACTCCTCTTCCAGC 59 6659 6678 293 531191 n/a n/aAAGTGTATTGCCTAGATTTC 37 6784 6803 294 531192 n/a n/aGAGTGCCATCTTCTCTGCAC 61 6968 6987 295 531193 n/a n/aTTATTCCCAGCTCTAAAATA 23 7274 7293 296 531194 n/a n/aCTCACAATTCTGTAAGGGAA 64 7596 7615 297 531195 n/a n/aATAAAATATATTAAGGCAAC 61 7846 7865 298 531196 n/a n/aTTGAGTCAGACATCCTGTGA 38 7996 8015 299 531197 n/a n/aTACCTTTTCTCCATGTCATT 42 8148 8167 300 531198 n/a n/aGGGATTTTGCTGAAGCTGGT 73 8273 8292 301 531199 n/a n/aCTTTGAATAGAAAATGACTA 1 8415 8434 302 531200 n/a n/a CAAAATCACAAGTTCTAGAT51 8617 8636 303 531201 n/a n/a TTTCCAATACTTTTACAAAT 52 8760 8779 304531202 n/a n/a ATTAATAAGCATCTCTCTGA 31 9109 9128 305 531203 n/a n/aTGACTATCCAATTTCTAGTT 67 9253 9272 306 531204 n/a n/aCTTGTAGTCTGCACTTAATG 60 9418 9437 307 531205 n/a n/aACATTTTTTAAGTACAGGAA 0 9602 9621 308 531206 n/a n/a GAAATGTCTAGCATTTTCTA28 9755 9774 309 531207 n/a n/a CCACTTATTTGATGACCACA 64 9915 9934 310531208 n/a n/a TCCAGAATACTGCCCCATCT 23 10050 10069 311 531209 n/a n/aTGGATTCATTTTCTGCAAAT 81 10175 10194 312 531210 n/a n/aAGACATTGTCAAATGTCCCC 60 10322 10341 313 531211 n/a n/aTTGATGTCAGCACTGTTGAC 77 10480 10499 314 531212 n/a n/aACATCAGTAGCTTCAGATGT 56 10618 10637 315 531213 n/a n/aCAAAATTAATTGTGCATAAT 13 10820 10839 316 531214 n/a n/aTTTTTCTTTAAATTTTGCTA 37 11120 11139 317 531215 n/a n/aTAGAGATTTTATGTACTTGG 63 11245 11264 318 531216 n/a n/aAAACACAGGAATTTGCAGAC 33 11408 11427 319 531217 n/a n/aGTGGAATAAACCATAATCTA 47 11579 11598 320 531218 n/a n/aGATAATTCTTTTCACAGACA 72 12028 12047 321 531219 n/a n/aCTTCTCTATCTCCCAGTGTT 61 12227 12246 322 531220 n/a n/aCAATACAGGTAAATTTCACG 56 12374 12393 323 531221 n/a n/aAAGGGATTTAAAATTTTTAT 0 12507 12526 324 531222 n/a n/aGGCAAGCTGTACAAGAAAAA 19 12642 12661 325 531223 n/a n/aTGTACTCACCGGTACTCTGC 58 12805 12824 326 531224 n/a n/aAAGAGAATGCTCAGAAATGG 25 13435 13454 327 531225 n/a n/aACACTTGTACCCCATACATC 45 13560 13579 328 531226 n/a n/aGACAGTAGAGACTGGGAAGG 12 13708 13727 329 531227 n/a n/aTACCAATTTCTGAAAGGGCA 72 14224 14243 330 531228 n/a n/aCAGAGTAAACTCCCCATCTC 33 14387 14406 331 531229 n/a n/aCTTCAAAGCCAGCAGTGTAA 69 14514 14533 332 531230 n/a n/aCTTACTGGGCTAAAATCAAG 46 14639 14658 333 531231 n/a n/aTATCACTGTACTAGTTTCCT 94 14744 14763 334 14815 14834 14886 14905 1494514964 15005 15024 15077 15096 15220 15239 15292 15311 15351 15370 1541115430 15483 15502 15555 15574 15613 15632 15685 15704 15815 15834 1588715906 15945 15964 531232 n/a n/a CTGTACTAGTTTCCTATAAC 85 14739 14758 33514810 14829 14881 14900 14940 14959 15000 15019 15072 15091 15215 1523415287 15306 15346 15365 15406 15425 15478 15497 15550 15569 15608 1562715680 15699 15810 15829 15882 15901 15940 15959 531233 n/a n/aACTGTACTAGTTTCCTATAA 86 14740 14759 336 14811 14830 14882 14901 1494114960 15001 15020 15073 15092 15216 15235 15288 15307 15347 15366 1540715426 15479 15498 15551 15570 15609 15628 15681 15700 15811 15830 1588315902 15941 15960 531234 n/a n/a CACTGTACTAGTTTCCTATA 86 14741 14760 33714812 14831 14883 14902 14942 14961 15002 15021 15074 15093 15217 1523615289 15308 15348 15367 15408 15427 15480 15499 15552 15571 15610 1562915682 15701 15812 15831 15884 15903 15942 15961 531235 n/a n/aTCACTGTACTAGTTTCCTAT 86 14742 14761 338 14813 14832 14884 14903 1494314962 15003 15022 15075 15094 15218 15237 15290 15309 15349 15368 1540915428 15481 15500 15553 15572 15611 15630 15683 15702 15813 15832 1588515904 15943 15962 531236 n/a n/a ATCACTGTACTAGTTTCCTA 87 14743 14762 33914814 14833 14885 14904 14944 14963 15004 15023 15076 15095 15219 1523815291 15310 15350 15369 15410 15429 15482 15501 15554 15573 15612 1563115684 15703 15814 15833 15886 15905 15944 15963 531237 n/a n/aGTGGAATGTCATGGCAATTT 56 16399 16418 340

Example 2: Antisense Inhibition of Human PKK in HepaRG™ Cells byAntisense Oligonucleotides with 2′-MOE Sugar Modifications

Additional antisense oligonucleotides were designed targeting a PKKnucleic acid and were tested for their effects on PKK mRNA in vitro.

The chimeric antisense oligonucleotides in the tables below weredesigned as 5-10-5 MOE gapmers, 4-9-4 MOE gapmers, 4-10-4 MOE gapmers,4-10-3 MOE gapmers, 3-10-4 MOE gapmers, or 3-10-3 MOE gapmers. The5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gapsegment comprises often 2′-deoxynucleosides and is flanked by wingsegments on the 5′ direction and the 3′ direction comprising fivenucleosides each. The 4-9-4 MOE gapmers are 17 nucleosides in length,wherein the central gap segment comprises of nine 2′-deoxynucleosidesand is flanked by wing segments on the 5′ direction and the 3′ directioncomprising four nucleosides each. The 4-10-4 MOE gapmers are 18nucleosides in length, wherein the central gap segment comprises often2′-deoxynucleosides and is flanked by wing segments on the 5′ directionand the 3′ direction comprising four nucleosides each. The 4-10-3 MOEgapmers are 17 nucleosides in length, wherein the central gap segmentcomprises often 2′-deoxynucleosides and is flanked by wing segments onthe 5′ direction and the 3′ direction comprising four and threenucleosides respectively. The 3-10-4 MOE gapmers are 17 nucleosides inlength, wherein the central gap segment comprises often2′-deoxynucleosides and is flanked by wing segments on the 5′ directionand the 3′ direction comprising three and four nucleosides respectively.The 3-10-3 MOE gapmers are 16 nucleosides in length, wherein the centralgap segment comprises often 2′-deoxynucleosides and is flanked by wingsegments on the 5′ direction and the 3′ direction comprising threenucleosides each. Each nucleoside in the 5′ wing segment and eachnucleoside in the 3′ wing segment has a 2′-O-methoxyethyl modification.The internucleoside linkages throughout each gapmer are phosphorothioatelinkages. All cytosine residues throughout each gapmer are5-methylcytosines. “Start site” indicates the 5′-most nucleoside towhich the gapmer is targeted in the human gene sequence. “Stop site”indicates the 3′-most nucleoside to which the gapmer is targeted in thehuman gene sequence. Each gapmer listed in the tables below is targetedto either SEQ ID NO: 1 or SEQ ID NO: 10. ‘n/a’ indicates that theantisense oligonucleotide does not target that particular gene sequence.

Cultured HepaRG™ cells at a density of 20,000 cells per well weretransfected using electroporation with 5,000 nM antisenseoligonucleotide. After a treatment period of approximately 24 hours, RNAwas isolated from the cells and PKK mRNA levels were measured byquantitative real-time PCR. Human primer probe set RTS3454 was used tomeasure mRNA levels. PKK mRNA levels were adjusted according to totalRNA content, as measured by RIBOGREEN®. The antisense oligonucleotideswere tested in a series of experiments that had similar cultureconditions. The results for each experiment are presented in separatetables shown below. Results are presented as percent inhibition of PKK,relative to untreated control cells.

TABLE 5 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 SEQ ISISStart Stop % Start Stop ID NO Site Site Sequence Motif inhibition SiteSite NO 531231 n/a n/a TATCACTGTACTAGTTTCCT 5-10-5 98 14744 14763 33414815 14834 14886 14905 14945 14964 15005 15024 15077 15096 15220 1523915292 15311 15351 15370 15411 15430 15483 15502 15555 15574 15613 1563215685 15704 15815 15834 15887 15906 15945 15964 546131 4 23ATGAACGGTCTTCAAGCTGT 5-10-5 75 3396 3415 341 547269 5 24AATGAACGGTCTTCAAGCTG 5-10-5 56 3397 3416 342 547270 7 26AAAATGAACGGTCTTCAAGC 5-10-5 68 3399 3418 343 547271 10 29TTAAAAATGAACGGTCTTCA 5-10-5 60 3402 3421 344 547272 13 32CACTTAAAAATGAACGGTCT 5-10-5 82 3405 3424 345 547273 25 44TGAGTCTCTTGTCACTTAAA 5-10-5 93 3417 3436 346 547274 29 48GAGGTGAGTCTCTTGTCACT 5-10-5 70 3421 3440 347 546136 30 49GGAGGTGAGTCTCTTGTCAC 5-10-5 86 3422 3441 348 547275 32 51TTGGAGGTGAGTCTCTTGTC 5-10-5 87 3424 3443 349 546137 40 59ATTGCTTCTTGGAGGTGAGT 5-10-5 76 3432 3451 350 547276 42 61CAATTGCTTCTTGGAGGTGA 5-10-5 93 3434 3453 351 547277 44 63CACAATTGCTTCTTGGAGGT 5-10-5 75 3436 3455 352 547278 45 64ACACAATTGCTTCTTGGAGG 5-10-5 70 3437 3456 353 546138 47 66AAACACAATTGCTTCTTGGA 5-10-5 69 3439 3458 354 547279 48 67AAAACACAATTGCTTCTTGG 5-10-5 69 3440 3459 355 547280 49 68GAAAACACAATTGCTTCTTG 5-10-5 47 3441 3460 356 547281 70 89TTGCTTGAATAAAATCATTC 5-10-5 41 4069 4088 357 546140 72 91GCTTGCTTGAATAAAATCAT 5-10-5 60 4071 4090 358 547282 74 93TTGCTTGCTTGAATAAAATC 5-10-5 53 4073 4092 359 547283 76 95AGTTGCTTGCTTGAATAAAA 5-10-5 67 4075 4094 360 546141 82 101GAAATAAGTTGCTTGCTTGA 5-10-5 56 4081 4100 361 547284 86 105AAATGAAATAAGTTGCTTGC 5-10-5 26 4085 4104 362 547285 102 121ACTGTAGCAAACAAGGAAAT 5-10-5 51 4101 4120 363 546143 106 125GGAAACTGTAGCAAACAAGG 5-10-5 46 4105 4124 364 546144 110 129CACAGGAAACTGTAGCAAAC 5-10-5 75 4109 4128 365 547286 117 136AGACATCCACAGGAAACTGT 5-10-5 68 n/a n/a 366 547287 120 139GTCAGACATCCACAGGAAAC 5-10-5 69 n/a n/a 367 546146 123 142TGAGTCAGACATCCACAGGA 5-10-5 72 n/a n/a 368 547288 131 150CATAGAGTTGAGTCAGACAT 5-10-5 80 8003 8022 369 546147 132 151TCATAGAGTTGAGTCAGACA 5-10-5 76 8004 8023 370 547289 133 152TTCATAGAGTTGAGTCAGAC 5-10-5 74 8005 8024 371 546148 137 156CGTTTTCATAGAGTTGAGTC 5-10-5 68 8009 8028 372 546149 155 174CCCCACCTCTGAAGAAGGCG 5-10-5 83 8027 8046 373 546150 158 177CATCCCCACCTCTGAAGAAG 5-10-5 58 8030 8049 374 547290 163 182AGCTACATCCCCACCTCTGA 5-10-5 76 8035 8054 375 546151 166 185GGAAGCTACATCCCCACCTC 5-10-5 76 8038 8057 376 547291 168 187ATGGAAGCTACATCCCCACC 5-10-5 74 8040 8059 377 547292 171 190TACATGGAAGCTACATCCCC 5-10-5 60 8043 8062 378 546152 172 191GTACATGGAAGCTACATCCC 5-10-5 73 8044 8063 379 546153 176 195GGGTGTACATGGAAGCTACA 5-10-5 76 8048 8067 380 546154 195 214TGGCAGTATTGGGCATTTGG 5-10-5 85 8067 8086 381 547293 199 218CATCTGGCAGTATTGGGCAT 5-10-5 92 8071 8090 382 547294 201 220CTCATCTGGCAGTATTGGGC 5-10-5 85 8073 8092 383 546155 202 221CCTCATCTGGCAGTATTGGG 5-10-5 47 8074 8093 384 547295 203 222ACCTCATCTGGCAGTATTGG 5-10-5 88 8075 8094 385 547296 206 225TGCACCTCATCTGGCAGTAT 5-10-5 72 8078 8097 386 546156 211 230GAATGTGCACCTCATCTGGC 5-10-5 81 8083 8102 387 547297 213 232TGGAATGTGCACCTCATCTG 5-10-5 84 8085 8104 388 546157 216 235GGGTGGAATGTGCACCTCAT 5-10-5 85 8088 8107 389 547298 218 237TTGGGTGGAATGTGCACCTC 5-10-5 90 8090 8109 390 546158 219 238CTTGGGTGGAATGTGCACCT 5-10-5 95 8091 8110 391 546159 229 248TAGCAAACACCTTGGGTGGA 5-10-5 76 8101 8120 392 546160 235 254ACTGAATAGCAAACACCTTG 5-10-5 78 8107 8126 393 547299 237 256AAACTGAATAGCAAACACCT 5-10-5 76 8109 8128 394 546163 250 269ACTTGCTGGAAGAAAACTGA 5-10-5 42 8122 8141 395 547300 252 271GAACTTGCTGGAAGAAAACT 5-10-5 37 8124 8143 396 546164 257 276TGATTGAACTTGCTGGAAGA 5-10-5 33 8129 8148 397 546165 260 279CATTGATTGAACTTGCTGGA 5-10-5 71 8132 8151 398 547301 261 280TCATTGATTGAACTTGCTGG 5-10-5 80 8133 8152 399 546166 263 282TGTCATTGATTGAACTTGCT 5-10-5 70 8135 8154 400 547302 266 285CCATGTCATTGATTGAACTT 5-10-5 58 8138 8157 401 546167 268 287CTCCATGTCATTGATTGAAC 5-10-5 73 8140 8159 402 547303 270 289TTCTCCATGTCATTGATTGA 5-10-5 72 8142 8161 403 547304 273 292CTTTTCTCCATGTCATTGAT 5-10-5 71 8145 8164 404 547305 280 299ACCAAACCTTTTCTCCATGT 5-10-5 47 n/a n/a 405 546170 283 302GCAACCAAACCTTTTCTCCA 5-10-5 54 n/a n/a 406 547306 284 303AGCAACCAAACCTTTTCTCC 5-10-5 62 n/a n/a 407 547307 286 305GAAGCAACCAAACCTTTTCT 5-10-5 58 n/a n/a 408 547308 290 309TCAAGAAGCAACCAAACCTT 5-10-5 66 n/a n/a 409 547309 293 312CTTTCAAGAAGCAACCAAAC 5-10-5 71 9827 9846 410 547310 295 314ATCTTTCAAGAAGCAACCAA 5-10-5 81 9829 9848 411 546171 297 316CTATCTTTCAAGAAGCAACC 5-10-5 81 9831 9850 412 547311 299 318CACTATCTTTCAAGAAGCAA 5-10-5 71 9833 9852 413 546172 301 320AACACTATCTTTCAAGAAGC 5-10-5 81 9835 9854 414 547312 325 344ATGTACTTTTGGCAGGGTTC 5-10-5 46 9859 9878 415 546173 327 346CGATGTACTTTTGGCAGGGT 5-10-5 84 9861 9880 416 547313 330 349GTTCGATGTACTTTTGGCAG 5-10-5 73 9864 9883 417

TABLE 6 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 SEQ ISISStart Stop % Start Stop ID NO Site Site Sequence Motif inhibition SiteSite NO 531231 n/a n/a TATCACTGTACTAGTTTCCT 5-10-5 86 14744 14763 33414815 14834 14886 14905 14945 14964 15005 15024 15077 15096 15220 1523915292 15311 15351 15370 15411 15430 15483 15502 15555 15574 15613 1563215685 15704 15815 15834 15887 15906 15945 15964 546174 333 352CCTGTTCGATGTACTTTTGG 5-10-5 74 9867 9886 418 547314 336 355GCACCTGTTCGATGTACTTT 5-10-5 73 9870 9889 419 546175 338 357CTGCACCTGTTCGATGTACT 5-10-5 78 9872 9891 420 547315 340 359AACTGCACCTGTTCGATGTA 5-10-5 50 9874 9893 421 547316 342 361GAAACTGCACCTGTTCGATG 5-10-5 75 9876 9895 422 547317 344 363CAGAAACTGCACCTGTTCGA 5-10-5 75 9878 9897 423 547318 345 364CCAGAAACTGCACCTGTTCG 5-10-5 74 9879 9898 424 546177 348 367TGTCCAGAAACTGCACCTGT 5-10-5 75 9882 9901 425 547319 351 370GAATGTCCAGAAACTGCACC 5-10-5 62 9885 9904 426 547320 353 372AGGAATGTCCAGAAACTGCA 5-10-5 73 9887 9906 427 547321 356 375TCAAGGAATGTCCAGAAACT 5-10-5 53 9890 9909 428 547322 358 377CTTCAAGGAATGTCCAGAAA 5-10-5 65 9892 9911 429 547323 361 380TTGCTTCAAGGAATGTCCAG 5-10-5 56 9895 9914 430 547324 363 382CATTGCTTCAAGGAATGTCC 5-10-5 76 9897 9916 431 547325 368 387GACCACATTGCTTCAAGGAA 5-10-5 67 9902 9921 432 546181 369 388TGACCACATTGCTTCAAGGA 5-10-5 75 9903 9922 433 547326 370 389ATGACCACATTGCTTCAAGG 5-10-5 48 9904 9923 434 547327 373 392TTGATGACCACATTGCTTCA 5-10-5 45 9907 9926 435 547328 375 394ATTTGATGACCACATTGCTT 5-10-5 40 9909 9928 436 547329 377 396TTATTTGATGACCACATTGC 5-10-5 24 9911 9930 437 547330 378 397CTTATTTGATGACCACATTG 5-10-5 60 9912 9931 438 546183 380 399CACTTATTTGATGACCACAT 5-10-5 69 9914 9933 439 547331 382 401AGCACTTATTTGATGACCAC 5-10-5 47 n/a n/a 440 546184 384 403CAAGCACTTATTTGATGACC 5-10-5 65 n/a n/a 441 547332 390 409CGATGGCAAGCACTTATTTG 5-10-5 44 n/a n/a 442 547333 395 414TGTCTCGATGGCAAGCACTT 5-10-5 76 n/a n/a 443 546186 396 415ATGTCTCGATGGCAAGCACT 5-10-5 84 n/a n/a 444 547334 397 416AATGTCTCGATGGCAAGCAC 5-10-5 74 n/a n/a 445 547335 402 421TTATAAATGTCTCGATGGCA 5-10-5 93 12658 12677 446 547336 403 422TTTATAAATGTCTCGATGGC 5-10-5 81 12659 12678 447 546188 407 426CTCCTTTATAAATGTCTCGA 5-10-5 95 12663 12682 448 547337 409 428AACTCCTTTATAAATGTCTC 5-10-5 84 12665 12684 449 547338 411 430TCAACTCCTTTATAAATGTC 5-10-5 71 12667 12686 450 547339 413 432TATCAACTCCTTTATAAATG 5-10-5 42 12669 12688 451 546190 419 438CTCTCATATCAACTCCTTTA 5-10-5 92 12675 12694 452 547340 422 441CTCCTCTCATATCAACTCCT 5-10-5 93 12678 12697 453 547341 424 443GACTCCTCTCATATCAACTC 5-10-5 87 12680 12699 454 546192 428 447AATTGACTCCTCTCATATCA 5-10-5 51 12684 12703 455 547342 433 452ATTAAAATTGACTCCTCTCA 5-10-5 66 12689 12708 456 546193 434 453CATTAAAATTGACTCCTCTC 5-10-5 57 12690 12709 457 547343 436 455CACATTAAAATTGACTCCTC 5-10-5 78 12692 12711 458 547344 438 457GACACATTAAAATTGACTCC 5-10-5 80 12694 12713 459 547345 439 458AGACACATTAAAATTGACTC 5-10-5 80 12695 12714 460 547346 444 463ACCTTAGACACATTAAAATT 5-10-5 57 12700 12719 461 546195 448 467GCTAACCTTAGACACATTAA 5-10-5 83 12704 12723 462 547347 451 470ACTGCTAACCTTAGACACAT 5-10-5 82 12707 12726 463 546196 452 471CACTGCTAACCTTAGACACA 5-10-5 83 12708 12727 464 547348 453 472ACACTGCTAACCTTAGACAC 5-10-5 83 12709 12728 465 547349 458 477CTTCAACACTGCTAACCTTA 5-10-5 88 12714 12733 466 546198 459 478TCTTCAACACTGCTAACCTT 5-10-5 85 12715 12734 467 547350 464 483GGCATTCTTCAACACTGCTA 5-10-5 96 12720 12739 468 546199 465 484TGGCATTCTTCAACACTGCT 5-10-5 97 12721 12740 469 547351 467 486TTTGGCATTCTTCAACACTG 5-10-5 92 12723 12742 470 546200 500 519AAAACTGGCAGCGAATGTTA 5-10-5 91 12756 12775 471 547352 541 560CCGGTACTCTGCCTTGTGAA 5-10-5 94 12797 12816 472 547354 547 566ATTGTTCCGGTACTCTGCCT 5-10-5 89 n/a n/a 473 546203 548 567AATTGTTCCGGTACTCTGCC 5-10-5 76 n/a n/a 474 547355 549 568CAATTGTTCCGGTACTCTGC 5-10-5 77 n/a n/a 475 546204 555 574AATAGGCAATTGTTCCGGTA 5-10-5 91 n/a n/a 476 547356 556 575TAATAGGCAATTGTTCCGGT 5-10-5 83 n/a n/a 477 547357 559 578CTTTAATAGGCAATTGTTCC 5-10-5 78 14130 14149 478 546205 562 581GTACTTTAATAGGCAATTGT 5-10-5 83 14133 14152 479 547359 569 588CGGGACTGTACTTTAATAGG 5-10-5 81 14140 14159 480 546208 605 624CGTTACTCAGCACCTTTATA 5-10-5 92 14176 14195 481 546209 629 648GCTTCAGTGAGAATCCAGAT 5-10-5 73 14200 14219 482 546210 651 670CCAATTTCTGAAAGGGCACA 5-10-5 79 14222 14241 483 547360 653 672AACCAATTTCTGAAAGGGCA 5-10-5 88 n/a n/a 484 547361 655 674GCAACCAATTTCTGAAAGGG 5-10-5 46 n/a n/a 485 546211 656 675GGCAACCAATTTCTGAAAGG 5-10-5 42 n/a n/a 486 546212 678 697AGATGCTGGAAGATGTTCAT 5-10-5 48 26126 26145 487 547362 701 720CAACATCCACATCTGAGAAC 5-10-5 47 26149 26168 488 547363 703 722GGCAACATCCACATCTGAGA 5-10-5 84 26151 26170 489 546213 707 726CCCTGGCAACATCCACATCT 5-10-5 82 26155 26174 490

TABLE 7 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 SEQ ISISStart Stop % Start Stop ID NO Site Site Sequence Motif inhibition SiteSite NO 531231 n/a n/a TATCACTGTACTAGTTTCCT 5-10-5 88 14744 14763 33414815 14834 14886 14905 14945 14964 15005 15024 15077 15096 15220 1523915292 15311 15351 15370 15411 15430 15483 15502 15555 15574 15613 1563215685 15704 15815 15834 15887 15906 15945 15964 547364 710 729GAACCCTGGCAACATCCACA 5-10-5 92 26158 26177 491 546214 712 731GAGAACCCTGGCAACATCCA 5-10-5 88 26160 26179 492 547365 713 732TGAGAACCCTGGCAACATCC 5-10-5 81 26161 26180 493 547366 717 736GGAGTGAGAACCCTGGCAAC 5-10-5 86 26165 26184 494 546216 719 738CTGGAGTGAGAACCCTGGCA 5-10-5 93 26167 26186 495 547367 721 740ATCTGGAGTGAGAACCCTGG 5-10-5 76 26169 26188 496 547368 723 742GCATCTGGAGTGAGAACCCT 5-10-5 89 26171 26190 497 547369 725 744AAGCATCTGGAGTGAGAACC 5-10-5 76 26173 26192 498 547370 728 747CAAAAGCATCTGGAGTGAGA 5-10-5 73 26176 26195 499 546217 730 749CACAAAAGCATCTGGAGTGA 5-10-5 83 26178 26197 500 546218 740 759TGGTCCGACACACAAAAGCA 5-10-5 71 26188 26207 501 547371 741 760ATGGTCCGACACACAAAAGC 5-10-5 66 26189 26208 502 547372 742 761GATGGTCCGACACACAAAAG 5-10-5 32 26190 26209 503 547373 745 764GCAGATGGTCCGACACACAA 5-10-5 90 26193 26212 504 546220 750 769TAGGTGCAGATGGTCCGACA 5-10-5 71 26198 26217 505 547374 752 771GATAGGTGCAGATGGTCCGA 5-10-5 81 26200 26219 506 547375 754 773GTGATAGGTGCAGATGGTCC 5-10-5 72 26202 26221 507 546222 756 775GGGTGATAGGTGCAGATGGT 5-10-5 12 26204 26223 508 547376 778 797GAATGTAAAGAAGAGGCAGT 5-10-5 43 26226 26245 509 546224 780 799TAGAATGTAAAGAAGAGGCA 5-10-5 65 26228 26247 510 547377 788 807CATTTGTATAGAATGTAAAG 5-10-5 6 26236 26255 511 547378 790 809TACATTTGTATAGAATGTAA 5-10-5 0 26238 26257 512 546226 793 812CCATACATTTGTATAGAATG 5-10-5 37 26241 26260 513 547379 802 821CTCGATTTTCCATACATTTG 5-10-5 37 26250 26269 514 547380 805 824TGACTCGATTTTCCATACAT 5-10-5 42 26253 26272 515 546228 806 825GTGACTCGATTTTCCATACA 5-10-5 60 26254 26273 516 547381 807 826TGTGACTCGATTTTCCATAC 5-10-5 49 26255 26274 517 547382 810 829CTTTGTGACTCGATTTTCCA 5-10-5 62 26258 26277 518 547383 812 831TTCTTTGTGACTCGATTTTC 5-10-5 37 n/a n/a 519 546229 816 835ACATTTCTTTGTGACTCGAT 5-10-5 19 n/a n/a 520 547384 818 837AAACATTTCTTTGTGACTCG 5-10-5 50 n/a n/a 521 547385 847 866TGTGCCACTTTCAGATGTTT 5-10-5 80 27111 27130 522 546230 848 867GTGTGCCACTTTCAGATGTT 5-10-5 70 27112 27131 523 546231 852 871CTTGGTGTGCCACTTTCAGA 5-10-5 79 27116 27135 524 547386 853 872ACTTGGTGTGCCACTTTCAG 5-10-5 78 27117 27136 525 546232 857 876AGGAACTTGGTGTGCCACTT 5-10-5 86 27121 27140 526 547387 878 897TGGTGTTTTCTTGAGGAGTA 5-10-5 73 27142 27161 527 546233 879 898ATGGTGTTTTCTTGAGGAGT 5-10-5 69 27143 27162 528 547388 880 899TATGGTGTTTTCTTGAGGAG 5-10-5 55 27144 27163 529 547389 884 903CAGATATGGTGTTTTCTTGA 5-10-5 61 27148 27167 530 546234 885 904CCAGATATGGTGTTTTCTTG 5-10-5 69 27149 27168 531 547390 887 906ATCCAGATATGGTGTTTTCT 5-10-5 63 27151 27170 532 547391 889 908ATATCCAGATATGGTGTTTT 5-10-5 32 27153 27172 533 546235 893 912GGCTATATCCAGATATGGTG 5-10-5 77 27157 27176 534 547392 895 914AAGGCTATATCCAGATATGG 5-10-5 81 27159 27178 535 546236 900 919GTTAAAAGGCTATATCCAGA 5-10-5 50 27164 27183 536 546237 903 922CAGGTTAAAAGGCTATATCC 5-10-5 64 27167 27186 537 547393 905 924TGCAGGTTAAAAGGCTATAT 5-10-5 73 27169 27188 538 547394 907 926TTTGCAGGTTAAAAGGCTAT 5-10-5 29 27171 27190 539 546238 909 928CTTTTGCAGGTTAAAAGGCT 5-10-5 63 27173 27192 540 546239 912 931GTTCTTTTGCAGGTTAAAAG 5-10-5 47 27176 27195 541 547395 914 933AAGTTCTTTTGCAGGTTAAA 5-10-5 15 27178 27197 542 546240 917 936GTAAAGTTCTTTTGCAGGTT 5-10-5 23 27181 27200 543 546241 920 939CAGGTAAAGTTCTTTTGCAG 5-10-5 69 27184 27203 544 547396 921 940TCAGGTAAAGTTCTTTTGCA 5-10-5 49 n/a n/a 545 547397 923 942GTTCAGGTAAAGTTCTTTTG 5-10-5 27 n/a n/a 546 546242 925 944GGGTTCAGGTAAAGTTCTTT 5-10-5 8 n/a n/a 547 547398 927 946CAGGGTTCAGGTAAAGTTCT 5-10-5 16 n/a n/a 548 547399 928 947GCAGGGTTCAGGTAAAGTTC 5-10-5 10 n/a n/a 549 547400 930 949TGGCAGGGTTCAGGTAAAGT 5-10-5 0 n/a n/a 550 547401 933 952GAATGGCAGGGTTCAGGTAA 5-10-5 22 n/a n/a 551 546243 934 953AGAATGGCAGGGTTCAGGTA 5-10-5 16 n/a n/a 552 547402 937 956TTTAGAATGGCAGGGTTCAG 5-10-5 59 n/a n/a 553 547403 939 958ATTTTAGAATGGCAGGGTTC 5-10-5 10 27361 27380 554 546244 942 961TAAATTTTAGAATGGCAGGG 5-10-5 27 27364 27383 555 547404 956 975AGTCAACTCCCGGGTAAATT 5-10-5 64 27378 27397 556 547405 959 978CAAAGTCAACTCCCGGGTAA 5-10-5 47 27381 27400 557 546247 960 979CCAAAGTCAACTCCCGGGTA 5-10-5 90 27382 27401 558 546248 963 982CCTCCAAAGTCAACTCCCGG 5-10-5 86 27385 27404 559 547406 965 984CTCCTCCAAAGTCAACTCCC 5-10-5 81 27387 27406 560 546249 968 987CTTCTCCTCCAAAGTCAACT 5-10-5 68 27390 27409 561 547407 975 994TTCAATTCTTCTCCTCCAAA 5-10-5 59 27397 27416 562 546250 977 996CATTCAATTCTTCTCCTCCA 5-10-5 65 27399 27418 563 547408 980 999TCACATTCAATTCTTCTCCT 5-10-5 84 27402 27421 564 547409 982 1001AGTCACATTCAATTCTTCTC 5-10-5 67 27404 27423 565 546251 1007 1026GGCAAACATTCACTCCTTTA 5-10-5 92 27429 27448 566

TABLE 8 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 SEQ ISISStart Stop % Start Stop ID NO Site Site Sequence Motif inhibition SiteSite NO 531231 n/a n/a TATCACTGTACTAGTTTCCT 5-10-5 95 14744 14763 34414815 14834 14886 14905 14945 14964 15005 15024 15077 15096 15220 1523915292 15311 15351 15370 15411 15430 15483 15502 15555 15574 15613 1563215685 15704 15815 15834 15887 15906 15945 15964 546252 1011 1030TCTTGGCAAACATTCACTCC 5-10-5 73 27433 27452 567 546253 1014 1033GTCTCTTGGCAAACATTCAC 5-10-5 98 27436 27455 568 547410 1017 1036CAAGTCTCTTGGCAAACATT 5-10-5 88 27439 27458 569 546254 1019 1038TGCAAGTCTCTTGGCAAACA 5-10-5 95 27441 27460 570 546255 1024 1043CTTTGTGCAAGTCTCTTGGC 5-10-5 92 27446 27465 571 547411 1027 1046CATCTTTGTGCAAGTCTCTT 5-10-5 79 27449 27468 572 546256 1028 1047TCATCTTTGTGCAAGTCTCT 5-10-5 83 27450 27469 573 547412 1029 1048ATCATCTTTGTGCAAGTCTC 5-10-5 73 27451 27470 574 546258 1036 1055ACAGCGAATCATCTTTGTGC 5-10-5 74 27458 27477 575 546259 1040 1059ACTGACAGCGAATCATCTTT 5-10-5 86 27462 27481 576 546260 1045 1064GAAAAACTGACAGCGAATCA 5-10-5 84 27467 27486 577 547413 1047 1066GTGAAAAACTGACAGCGAAT 5-10-5 94 27469 27488 578 546263 1061 1080GGAGTAAAGAATAAGTGAAA 5-10-5 0 27483 27502 579 547414 1063 1082TGGGAGTAAAGAATAAGTGA 5-10-5 76 27485 27504 580 547415 1065 1084TCTGGGAGTAAAGAATAAGT 5-10-5 71 27487 27506 581 546265 1069 1088GTCTTCTGGGAGTAAAGAAT 5-10-5 65 27491 27510 582 546266 1072 1091ACAGTCTTCTGGGAGTAAAG 5-10-5 63 27494 27513 583 547416 1075 1094CTTACAGTCTTCTGGGAGTA 5-10-5 79 27497 27516 584 546267 1076 1095CCTTACAGTCTTCTGGGAGT 5-10-5 72 27498 27517 585 547417 1077 1096TCCTTACAGTCTTCTGGGAG 5-10-5 68 27499 27518 586 546268 1079 1098CTTCCTTACAGTCTTCTGGG 5-10-5 93 27501 27520 587 547418 1092 1111CACTTACACTTCTCTTCCTT 5-10-5 0 n/a n/a 588 546270 1093 1112ACACTTACACTTCTCTTCCT 5-10-5 32 n/a n/a 589 546271 1097 1116AGAAACACTTACACTTCTCT 5-10-5 60 n/a n/a 590 547419 1101 1120CTTAAGAAACACTTACACTT 5-10-5 51 n/a n/a 591 547420 1112 1131CCATAGATAATCTTAAGAAA 5-10-5 8 27633 27652 592 547421 1115 1134CATCCATAGATAATCTTAAG 5-10-5 69 27636 27655 593 547422 1117 1136ACCATCCATAGATAATCTTA 5-10-5 70 27638 27657 594 546275 1119 1138GAACCATCCATAGATAATCT 5-10-5 87 27640 27659 595 546276 1123 1142TGGAGAACCATCCATAGATA 5-10-5 74 27644 27663 596 546277 1146 1165TGTGTCCCATACGCAATCCT 5-10-5 90 27667 27686 597 547423 1150 1169CCCTTGTGTCCCATACGCAA 5-10-5 95 27671 27690 598 546279 1153 1172GCTCCCTTGTGTCCCATACG 5-10-5 82 27674 27693 599 547424 1156 1175AGAGCTCCCTTGTGTCCCAT 5-10-5 90 27677 27696 600 546280 1158 1177CCAGAGCTCCCTTGTGTCCC 5-10-5 86 27679 27698 601 547425 1161 1180TAACCAGAGCTCCCTTGTGT 5-10-5 85 27682 27701 602 546281 1162 1181GTAACCAGAGCTCCCTTGTG 5-10-5 85 27683 27702 603 547426 1164 1183GAGTAACCAGAGCTCCCTTG 5-10-5 92 27685 27704 604 547427 1166 1185AAGAGTAACCAGAGCTCCCT 5-10-5 79 27687 27706 605 547428 1169 1188TCAAAGAGTAACCAGAGCTC 5-10-5 78 27690 27709 606 546283 1171 1190TCTCAAAGAGTAACCAGAGC 5-10-5 88 27692 27711 607 547429 1173 1192AATCTCAAAGAGTAACCAGA 5-10-5 81 27694 27713 608 547430 1174 1193CAATCTCAAAGAGTAACCAG 5-10-5 70 27695 27714 609 546284 1176 1195CACAATCTCAAAGAGTAACC 5-10-5 89 27697 27716 610 546285 1180 1199GTTACACAATCTCAAAGAGT 5-10-5 76 27701 27720 611 547431 1184 1203CAGTGTTACACAATCTCAAA 5-10-5 67 27705 27724 612 547432 1186 1205CCCAGTGTTACACAATCTCA 5-10-5 90 27707 27726 613 547433 1189 1208GTCCCCAGTGTTACACAATC 5-10-5 63 27710 27729 614 546287 1192 1211GTTGTCCCCAGTGTTACACA 5-10-5 82 27713 27732 615 546288 1240 1259GTTTGTTCCTCCAACAATGC 5-10-5 78 27916 27935 616 547434 1243 1262AGAGTTTGTTCCTCCAACAA 5-10-5 54 27919 27938 617 547435 1248 1267CAAGAAGAGTTTGTTCCTCC 5-10-5 85 27924 27943 618 546290 1251 1270CCCCAAGAAGAGTTTGTTCC 5-10-5 86 27927 27946 619 547436 1253 1272CTCCCCAAGAAGAGTTTGTT 5-10-5 0 27929 27948 620 547437 1255 1274CTCTCCCCAAGAAGAGTTTG 5-10-5 50 27931 27950 621 547438 1261 1280GGGCCACTCTCCCCAAGAAG 5-10-5 82 27937 27956 622 546291 1263 1282CAGGGCCACTCTCCCCAAGA 5-10-5 81 27939 27958 623 547439 1298 1317TCTGAGCTGTCAGCTTCACC 5-10-5 85 27974 27993 624 546293 1301 1320GCCTCTGAGCTGTCAGCTTC 5-10-5 64 27977 27996 625 547440 1327 1346TCCTATGAGTGACCCTCCAC 5-10-5 67 28003 28022 626 546294 1328 1347GTCCTATGAGTGACCCTCCA 5-10-5 72 28004 28023 627 547441 1331 1350GGTGTCCTATGAGTGACCCT 5-10-5 62 28007 28026 628 547442 1332 1351TGGTGTCCTATGAGTGACCC 5-10-5 42 28008 28027 629 547443 1336 1355CCACTGGTGTCCTATGAGTG 5-10-5 70 28012 28031 630 546295 1337 1356CCCACTGGTGTCCTATGAGT 5-10-5 67 28013 28032 631 546296 1370 1389GAAGCCCATCAAAGCAGTGG 5-10-5 27 n/a n/a 632 546297 1397 1416TATAGATGCGCCAAACATCC 5-10-5 82 30475 30494 633 547444 1398 1417CTATAGATGCGCCAAACATC 5-10-5 71 30476 30495 634 547445 1402 1421GCCACTATAGATGCGCCAAA 5-10-5 97 30480 30499 635 546299 1404 1423ATGCCACTATAGATGCGCCA 5-10-5 84 30482 30501 636 546300 1424 1443TAATGTCTGACAGATTTAAA 5-10-5 58 30502 30521 637 546301 1427 1446TTGTAATGTCTGACAGATTT 5-10-5 93 30505 30524 638 546302 1444 1463TGAGAAAGGTGTATCTTTTG 5-10-5 87 30522 30541 639 547446 1447 1466TTGTGAGAAAGGTGTATCTT 5-10-5 84 30525 30544 640 546303 1448 1467TTTGTGAGAAAGGTGTATCT 5-10-5 77 30526 30545 641 547447 1449 1468ATTTGTGAGAAAGGTGTATC 5-10-5 80 30527 30546 642

TABLE 9 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 SEQ ISISStart Stop % Start Stop ID NO Site Site Sequence Motif inhibition SiteSite NO 531231 n/a n/a TATCACTGTACTAGTTTCCT 5-10-5 96 14744 14763 33414815 14834 14886 14905 14945 14964 15005 15024 15077 15096 15220 1523915292 15311 15351 15370 15411 15430 15483 15502 15555 15574 15613 1563215685 15704 15815 15834 15887 15906 15945 15964 547448 1451 1470TTATTTGTGAGAAAGGTGTA 5-10-5 75 30529 30548 643 547449 1453 1472TTTTATTTGTGAGAAAGGTG 5-10-5 71 30531 30550 644 546304 1454 1473CTTTTATTTGTGAGAAAGGT 5-10-5 94 30532 30551 645 547450 1456 1475CTCTTTTATTTGTGAGAAAG 5-10-5 71 30534 30553 646 547451 1471 1490TTGGTGAATAATAATCTCTT 5-10-5 75 30549 30568 647 546306 1472 1491TTTGGTGAATAATAATCTCT 5-10-5 65 30550 30569 648 547452 1474 1493GTTTTGGTGAATAATAATCT 5-10-5 47 30552 30571 649 546307 1478 1497TATAGTTTTGGTGAATAATA 5-10-5 12 30556 30575 650 546308 1482 1501ACTTTATAGTTTTGGTGAAT 5-10-5 57 30560 30579 651 546309 1492 1511CCCTTCTGAGACTTTATAGT 5-10-5 88 30570 30589 652 546310 1496 1515GATTCCCTTCTGAGACTTTA 5-10-5 78 30574 30593 653 546311 1499 1518CATGATTCCCTTCTGAGACT 5-10-5 79 30577 30596 654 547453 1500 1519TCATGATTCCCTTCTGAGAC 5-10-5 81 30578 30597 655 547454 1502 1521TATCATGATTCCCTTCTGAG 5-10-5 92 30580 30599 656 547455 1503 1522ATATCATGATTCCCTTCTGA 5-10-5 88 30581 30600 657 547456 1506 1525GCGATATCATGATTCCCTTC 5-10-5 89 30584 30603 658 546313 1507 1526GGCGATATCATGATTCCCTT 5-10-5 60 30585 30604 659 547457 1509 1528AAGGCGATATCATGATTCCC 5-10-5 89 30587 30606 660 547458 1513 1532TATCAAGGCGATATCATGAT 5-10-5 84 30591 30610 661 547459 1519 1538GAGTTTTATCAAGGCGATAT 5-10-5 28 30597 30616 662 547460 1522 1541CTGGAGTTTTATCAAGGCGA 5-10-5 72 30600 30619 663 546316 1524 1543GCCTGGAGTTTTATCAAGGC 5-10-5 51 30602 30621 664 546317 1528 1547AGGAGCCTGGAGTTTTATCA 5-10-5 12 30606 30625 665 546318 1534 1553ATTCAAAGGAGCCTGGAGTT 5-10-5 47 30612 30631 666 547461 1537 1556GTAATTCAAAGGAGCCTGGA 5-10-5 49 30615 30634 667 547462 1539 1558GTGTAATTCAAAGGAGCCTG 5-10-5 59 30617 30636 668 546319 1541 1560CAGTGTAATTCAAAGGAGCC 5-10-5 50 30619 30638 669 547463 1564 1583TAGGCATATTGGTTTTTGGA 5-10-5 74 31870 31889 670 546320 1566 1585GGTAGGCATATTGGTTTTTG 5-10-5 72 31872 31891 671 546321 1569 1588GAAGGTAGGCATATTGGTTT 5-10-5 53 31875 31894 672 546322 1584 1603CTTGTGTCACCTTTGGAAGG 5-10-5 74 31890 31909 673 547464 1585 1604GCTTGTGTCACCTTTGGAAG 5-10-5 95 31891 31910 674 546323 1587 1606GTGCTTGTGTCACCTTTGGA 5-10-5 94 31893 31912 675 547465 1592 1611AAATTGTGCTTGTGTCACCT 5-10-5 88 31898 31917 676 547466 1596 1615GTATAAATTGTGCTTGTGTC 5-10-5 82 31902 31921 677 546324 1597 1616GGTATAAATTGTGCTTGTGT 5-10-5 73 31903 31922 678 547467 1598 1617TGGTATAAATTGTGCTTGTG 5-10-5 80 31904 31923 679 547468 1600 1619GTTGGTATAAATTGTGCTTG 5-10-5 61 31906 31925 680 546325 1602 1621CAGTTGGTATAAATTGTGCT 5-10-5 74 31908 31927 681 546326 1607 1626CCCAACAGTTGGTATAAATT 5-10-5 62 31913 31932 682 547469 1610 1629TTACCCAACAGTTGGTATAA 5-10-5 67 31916 31935 683 546327 1612 1631GGTTACCCAACAGTTGGTAT 5-10-5 95 31918 31937 684 546328 1624 1643GAAGCCCCATCCGGTTACCC 5-10-5 84 31930 31949 685 547470 1628 1647TCGAGAAGCCCCATCCGGTT 5-10-5 70 31934 31953 686 546329 1631 1650CCTTCGAGAAGCCCCATCCG 5-10-5 18 31937 31956 687 546330 1636 1655TTTCTCCTTCGAGAAGCCCC 5-10-5 55 31942 31961 688 547471 1638 1657CCTTTCTCCTTCGAGAAGCC 5-10-5 58 31944 31963 689 547472 1641 1660TCACCTTTCTCCTTCGAGAA 5-10-5 44 n/a n/a 690 546331 1642 1661TTCACCTTTCTCCTTCGAGA 5-10-5 59 n/a n/a 691 547473 1649 1668TTTGGATTTCACCTTTCTCC 5-10-5 5 n/a n/a 692 547474 1659 1678TGTAGAATATTTTGGATTTC 5-10-5 51 33103 33122 693 547475 1686 1705TTTGTTACCAAAGGAATATT 5-10-5 44 33130 33149 694 547476 1688 1707CATTTGTTACCAAAGGAATA 5-10-5 75 33132 33151 695 546336 1689 1708TCATTTGTTACCAAAGGAAT 5-10-5 66 33133 33152 696 547477 1692 1711TCTTCATTTGTTACCAAAGG 5-10-5 74 33136 33155 697 547478 1695 1714CATTCTTCATTTGTTACCAA 5-10-5 85 33139 33158 698 546339 1712 1731CTTGATATCTTTTCTGGCAT 5-10-5 65 33156 33175 699 546340 1716 1735TAATCTTGATATCTTTTCTG 5-10-5 30 33160 33179 700 547479 1718 1737TATAATCTTGATATCTTTTC 5-10-5 48 33162 33181 701 547480 1756 1775TTCTTTATAGCCAGCACAGA 5-10-5 60 33200 33219 702 547481 1758 1777CCTTCTTTATAGCCAGCACA 5-10-5 71 33202 33221 703 547482 1760 1779CCCCTTCTTTATAGCCAGCA 5-10-5 90 33204 33223 704 546343 1761 1780CCCCCTTCTTTATAGCCAGC 5-10-5 97 33205 33224 705 547483 1762 1781TCCCCCTTCTTTATAGCCAG 5-10-5 71 33206 33225 706 546345 1773 1792CAAGCATCTTTTCCCCCTTC 5-10-5 86 33217 33236 707 546346 1796 1815AGGGACCACCTGAATCTCCC 5-10-5 83 33895 33914 708 547484 1799 1818CTAAGGGACCACCTGAATCT 5-10-5 69 33898 33917 709 546347 1800 1819ACTAAGGGACCACCTGAATC 5-10-5 28 33899 33918 710 547485 1803 1822CAAACTAAGGGACCACCTGA 5-10-5 49 33902 33921 711 546348 1804 1823GCAAACTAAGGGACCACCTG 5-10-5 79 33903 33922 712 547486 1805 1824TGCAAACTAAGGGACCACCT 5-10-5 89 33904 33923 713 546349 1810 1829GTGTTTGCAAACTAAGGGAC 5-10-5 48 33909 33928 714 547487 1811 1830TGTGTTTGCAAACTAAGGGA 5-10-5 72 33910 33929 715 546350 1868 1887CCCTGCGGGCACAGCCTTCA 5-10-5 88 33967 33986 716 546351 1873 1892TTGCTCCCTGCGGGCACAGC 5-10-5 82 33972 33991 717 546352 1880 1899CACCAGGTTGCTCCCTGCGG 5-10-5 75 33979 33998 718 547488 1881 1900ACACCAGGTTGCTCCCTGCG 5-10-5 71 33980 33999 719

TABLE 10 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 SEQ ISISStart Stop % Start Stop ID NO Site Site Sequence Motif inhibition SiteSite NO 531231 n/a n/a TATCACTGTACTAGTTTCCT 5-10-5 72 14744 14763 33414815 14834 14886 14905 14945 14964 15005 15024 15077 15096 15220 1523915292 15311 15351 15370 15411 15430 15483 15502 15555 15574 15613 1563215685 15704 15815 15834 15887 15906 15945 15964 547448 1451 1470TTATTTGTGAGAAAGGTGTA 5-10-5 83 30529 30548 643 547449 1453 1472TTTTATTTGTGAGAAAGGTG 5-10-5 73 30531 30550 644 546304 1454 1473CTTTTATTTGTGAGAAAGGT 5-10-5 86 30532 30551 645 547450 1456 1475CTCTTTTATTTGTGAGAAAG 5-10-5 67 30534 30553 646 547451 1471 1490TTGGTGAATAATAATCTCTT 5-10-5 64 30549 30568 647 546306 1472 1491TTTGGTGAATAATAATCTCT 5-10-5 71 30550 30569 648 547452 1474 1493GTTTTGGTGAATAATAATCT 5-10-5 62 30552 30571 649 546307 1478 1497TATAGTTTTGGTGAATAATA 5-10-5 0 30556 30575 650 546308 1482 1501ACTTTATAGTTTTGGTGAAT 5-10-5 43 30560 30579 651 546309 1492 1511CCCTTCTGAGACTTTATAGT 5-10-5 81 30570 30589 652 546310 1496 1515GATTCCCTTCTGAGACTTTA 5-10-5 67 30574 30593 653 546311 1499 1518CATGATTCCCTTCTGAGACT 5-10-5 76 30577 30596 654 547453 1500 1519TCATGATTCCCTTCTGAGAC 5-10-5 81 30578 30597 655 547454 1502 1521TATCATGATTCCCTTCTGAG 5-10-5 78 30580 30599 656 547455 1503 1522ATATCATGATTCCCTTCTGA 5-10-5 66 30581 30600 657 547456 1506 1525GCGATATCATGATTCCCTTC 5-10-5 96 30584 30603 658 546313 1507 1526GGCGATATCATGATTCCCTT 5-10-5 75 30585 30604 659 547457 1509 1528AAGGCGATATCATGATTCCC 5-10-5 92 30587 30606 660 547458 1513 1532TATCAAGGCGATATCATGAT 5-10-5 64 30591 30610 661 547459 1519 1538GAGTTTTATCAAGGCGATAT 5-10-5 51 30597 30616 662 547460 1522 1541CTGGAGTTTTATCAAGGCGA 5-10-5 75 30600 30619 663 546316 1524 1543GCCTGGAGTTTTATCAAGGC 5-10-5 60 30602 30621 664 546317 1528 1547AGGAGCCTGGAGTTTTATCA 5-10-5 31 30606 30625 665 546318 1534 1553ATTCAAAGGAGCCTGGAGTT 5-10-5 46 30612 30631 666 547461 1537 1556GTAATTCAAAGGAGCCTGGA 5-10-5 55 30615 30634 667 547462 1539 1558GTGTAATTCAAAGGAGCCTG 5-10-5 54 30617 30636 668 546319 1541 1560CAGTGTAATTCAAAGGAGCC 5-10-5 61 30619 30638 669 547463 1564 1583TAGGCATATTGGTTTTTGGA 5-10-5 84 31870 31889 670 546320 1566 1585GGTAGGCATATTGGTTTTTG 5-10-5 69 31872 31891 671 546321 1569 1588GAAGGTAGGCATATTGGTTT 5-10-5 56 31875 31894 672 546322 1584 1603CTTGTGTCACCTTTGGAAGG 5-10-5 68 31890 31909 673 547464 1585 1604GCTTGTGTCACCTTTGGAAG 5-10-5 84 31891 31910 674 546323 1587 1606GTGCTTGTGTCACCTTTGGA 5-10-5 80 31893 31912 675 547465 1592 1611AAATTGTGCTTGTGTCACCT 5-10-5 85 31898 31917 676 547466 1596 1615GTATAAATTGTGCTTGTGTC 5-10-5 43 31902 31921 677 546324 1597 1616GGTATAAATTGTGCTTGTGT 5-10-5 82 31903 31922 678 547467 1598 1617TGGTATAAATTGTGCTTGTG 5-10-5 65 31904 31923 679 547468 1600 1619GTTGGTATAAATTGTGCTTG 5-10-5 46 31906 31925 680 546325 1602 1621CAGTTGGTATAAATTGTGCT 5-10-5 79 31908 31927 681 546326 1607 1626CCCAACAGTTGGTATAAATT 5-10-5 64 31913 31932 682 547469 1610 1629TTACCCAACAGTTGGTATAA 5-10-5 50 31916 31935 683 546327 1612 1631GGTTACCCAACAGTTGGTAT 5-10-5 84 31918 31937 684 546328 1624 1643GAAGCCCCATCCGGTTACCC 5-10-5 81 31930 31949 685 547470 1628 1647TCGAGAAGCCCCATCCGGTT 5-10-5 68 31934 31953 686 546329 1631 1650CCTTCGAGAAGCCCCATCCG 5-10-5 8 31937 31956 687 546330 1636 1655TTTCTCCTTCGAGAAGCCCC 5-10-5 67 31942 31961 688 547471 1638 1657CCTTTCTCCTTCGAGAAGCC 5-10-5 43 31944 31963 689 547472 1641 1660TCACCTTTCTCCTTCGAGAA 5-10-5 42 n/a n/a 690 546331 1642 1661TTCACCTTTCTCCTTCGAGA 5-10-5 44 n/a n/a 691 547473 1649 1668TTTGGATTTCACCTTTCTCC 5-10-5 26 n/a n/a 692 547474 1659 1678TGTAGAATATTTTGGATTTC 5-10-5 34 33103 33122 693 547475 1686 1705TTTGTTACCAAAGGAATATT 5-10-5 42 33130 33149 694 547476 1688 1707CATTTGTTACCAAAGGAATA 5-10-5 71 33132 33151 695 546336 1689 1708TCATTTGTTACCAAAGGAAT 5-10-5 73 33133 33152 696 547477 1692 1711TCTTCATTTGTTACCAAAGG 5-10-5 68 33136 33155 697 547478 1695 1714CATTCTTCATTTGTTACCAA 5-10-5 55 33139 33158 698 546339 1712 1731CTTGATATCTTTTCTGGCAT 5-10-5 64 33156 33175 699 546340 1716 1735TAATCTTGATATCTTTTCTG 5-10-5 56 33160 33179 700 547479 1718 1737TATAATCTTGATATCTTTTC 5-10-5 9 33162 33181 701 547480 1756 1775TTCTTTATAGCCAGCACAGA 5-10-5 49 33200 33219 702 547481 1758 1777CCTTCTTTATAGCCAGCACA 5-10-5 77 33202 33221 703 547482 1760 1779CCCCTTCTTTATAGCCAGCA 5-10-5 65 33204 33223 704 546343 1761 1780CCCCCTTCTTTATAGCCAGC 5-10-5 91 33205 33224 705 547483 1762 1781TCCCCCTTCTTTATAGCCAG 5-10-5 77 33206 33225 706 546345 1773 1792CAAGCATCTTTTCCCCCTTC 5-10-5 80 33217 33236 707 546346 1796 1815AGGGACCACCTGAATCTCCC 5-10-5 70 33895 33914 708 547484 1799 1818CTAAGGGACCACCTGAATCT 5-10-5 64 33898 33917 709 546347 1800 1819ACTAAGGGACCACCTGAATC 5-10-5 22 33899 33918 710 547485 1803 1822CAAACTAAGGGACCACCTGA 5-10-5 66 33902 33921 711 546348 1804 1823GCAAACTAAGGGACCACCTG 5-10-5 76 33903 33922 712 547486 1805 1824TGCAAACTAAGGGACCACCT 5-10-5 78 33904 33923 713 546349 1810 1829GTGTTTGCAAACTAAGGGAC 5-10-5 35 33909 33928 714 547487 1811 1830TGTGTTTGCAAACTAAGGGA 5-10-5 61 33910 33929 715 546350 1868 1887CCCTGCGGGCACAGCCTTCA 5-10-5 74 33967 33986 716 546351 1873 1892TTGCTCCCTGCGGGCACAGC 5-10-5 60 33972 33991 717 546352 1880 1899CACCAGGTTGCTCCCTGCGG 5-10-5 74 33979 33998 718 547488 1881 1900ACACCAGGTTGCTCCCTGCG 5-10-5 72 33980 33999 719

TABLE 11 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 SEQ ISISStart Stop % Start Stop ID NO Site Site Sequence Motif inhibition SiteSite NO 531231 n/a n/a TATCACTGTACTAGTTTCCT 5-10-5 90 14744 14763 33414815 14834 14886 14905 14945 14964 15005 15024 15077 15096 15220 1523915292 15311 15351 15370 15411 15430 15483 15502 15555 15574 15613 1563215685 15704 15815 15834 15887 15906 15945 15964 547489 1883 1902AGACACCAGGTTGCTCCCTG 5-10-5 34 33982 34001 720 547490 1885 1904GTAGACACCAGGTTGCTCCC 5-10-5 55 33984 34003 721 546353 1900 1919CTCAGCGACTTTGGTGTAGA 5-10-5 55 33999 34018 722 546354 1903 1922GTACTCAGCGACTTTGGTGT 5-10-5 47 34002 34021 723 547491 1906 1925CATGTACTCAGCGACTTTGG 5-10-5 47 34005 34024 724 547492 1911 1930CAGTCCATGTACTCAGCGAC 5-10-5 62 34010 34029 725 546356 1913 1932TCCAGTCCATGTACTCAGCG 5-10-5 60 34012 34031 726 546357 1947 1966GCTTTTCCATCACTGCTCTG 5-10-5 79 34046 34065 727 546358 1951 1970CTGAGCTTTTCCATCACTGC 5-10-5 83 34050 34069 728 547493 1952 1971TCTGAGCTTTTCCATCACTG 5-10-5 72 34051 34070 729 546359 1955 1974GCATCTGAGCTTTTCCATCA 5-10-5 79 34054 34073 730 546360 1958 1977ACTGCATCTGAGCTTTTCCA 5-10-5 13 34057 34076 731 547494 1963 1982TGGTGACTGCATCTGAGCTT 5-10-5 70 34062 34081 732 547495 1965 1984GCTGGTGACTGCATCTGAGC 5-10-5 61 34064 34083 733 547496 1967 1986ATGCTGGTGACTGCATCTGA 5-10-5 80 34066 34085 734 546362 1969 1988TCATGCTGGTGACTGCATCT 5-10-5 71 34068 34087 735 546363 1973 1992CTTCTCATGCTGGTGACTGC 5-10-5 81 34072 34091 736 547497 1977 1996ACTGCTTCTCATGCTGGTGA 5-10-5 68 34076 34095 737 546364 1979 1998GGACTGCTTCTCATGCTGGT 5-10-5 61 34078 34097 738 547498 1981 2000CTGGACTGCTTCTCATGCTG 5-10-5 44 34080 34099 739 547499 1983 2002CTCTGGACTGCTTCTCATGC 5-10-5 65 34082 34101 740 546365 1986 2005AGACTCTGGACTGCTTCTCA 5-10-5 64 34085 34104 741 547500 1989 2008CCTAGACTCTGGACTGCTTC 5-10-5 65 34088 34107 742 546366 1991 2010TGCCTAGACTCTGGACTGCT 5-10-5 79 34090 34109 743 547501 1993 2012ATTGCCTAGACTCTGGACTG 5-10-5 55 34092 34111 744 546367 1997 2016AAAAATTGCCTAGACTCTGG 5-10-5 61 34096 34115 745 546368 2003 2022GGTTGTAAAAATTGCCTAGA 5-10-5 44 34102 34121 746 547502 2006 2025TCAGGTTGTAAAAATTGCCT 5-10-5 64 34105 34124 747 546369 2007 2026CTCAGGTTGTAAAAATTGCC 5-10-5 51 34106 34125 748 547503 2008 2027ACTCAGGTTGTAAAAATTGC 5-10-5 66 34107 34126 749 547504 2010 2029GAACTCAGGTTGTAAAAATT 5-10-5 37 34109 34128 750 546370 2014 2033ACTTGAACTCAGGTTGTAAA 5-10-5 34 34113 34132 751 547505 2015 2034GACTTGAACTCAGGTTGTAA 5-10-5 69 34114 34133 752 546372 2021 2040GAATTTGACTTGAACTCAGG 5-10-5 49 34120 34139 753 546373 2025 2044CTCAGAATTTGACTTGAACT 5-10-5 59 34124 34143 754 547506 2028 2047AGGCTCAGAATTTGACTTGA 5-10-5 78 34127 34146 755 547507 2029 2048CAGGCTCAGAATTTGACTTG 5-10-5 56 34128 34147 756 546374 2030 2049CCAGGCTCAGAATTTGACTT 5-10-5 50 34129 34148 757 547508 2032 2051CCCCAGGCTCAGAATTTGAC 5-10-5 69 34131 34150 758 547509 2034 2053CCCCCCAGGCTCAGAATTTG 5-10-5 58 34133 34152 759 546375 2036 2055GACCCCCCAGGCTCAGAATT 5-10-5 48 34135 34154 760 547510 2041 2060ATGAGGACCCCCCAGGCTCA 5-10-5 40 34140 34159 761 547511 2042 2061GATGAGGACCCCCCAGGCTC 5-10-5 53 34141 34160 762 547512 2045 2064GCAGATGAGGACCCCCCAGG 5-10-5 74 34144 34163 763 547513 2046 2065TGCAGATGAGGACCCCCCAG 5-10-5 72 34145 34164 764 546378 2048 2067TTTGCAGATGAGGACCCCCC 5-10-5 79 34147 34166 765 546379 2056 2075CTCCATGCTTTGCAGATGAG 5-10-5 69 34155 34174 766 546380 2062 2081GCCACTCTCCATGCTTTGCA 5-10-5 81 34161 34180 767 547514 2066 2085AGATGCCACTCTCCATGCTT 5-10-5 85 34165 34184 768 546381 2068 2087GAAGATGCCACTCTCCATGC 5-10-5 73 34167 34186 769 547515 2069 2088AGAAGATGCCACTCTCCATG 5-10-5 58 34168 34187 770 546382 2072 2091CAAAGAAGATGCCACTCTCC 5-10-5 58 34171 34190 771 547516 2076 2095GATGCAAAGAAGATGCCACT 5-10-5 48 34175 34194 772 546383 2077 2096GGATGCAAAGAAGATGCCAC 5-10-5 57 34176 34195 773 547517 2079 2098TAGGATGCAAAGAAGATGCC 5-10-5 57 34178 34197 774 547518 2083 2102TCCTTAGGATGCAAAGAAGA 5-10-5 51 34182 34201 775 546384 2085 2104CGTCCTTAGGATGCAAAGAA 5-10-5 81 34184 34203 776 546385 2120 2139ATTGTCCTCAGCAGCTCTGA 5-10-5 67 34219 34238 777 547519 n/a n/aCCAGACATTGTCCTCAGCAG 5-10-5 76 34225 34244 778 546386 n/a n/aAGCCAGACATTGTCCTCAGC 5-10-5 78 34227 34246 779 547520 n/a n/aTCAGCCAGACATTGTCCTCA 5-10-5 76 34229 34248 780 547521 n/a n/aCTTCAGCCAGACATTGTCCT 5-10-5 58 34231 34250 781 546387 n/a n/aAGCGGGCTTCAGCCAGACAT 5-10-5 77 34237 34256 782 547522 n/a n/aGAAAGCGGGCTTCAGCCAGA 5-10-5 73 34240 34259 783 546388 n/a n/aCTGAAAGCGGGCTTCAGCCA 5-10-5 71 34242 34261 784 546389 2147 2166CGTGCTGAAAGCGGGCTTCA 5-10-5 71 34246 34265 785 546390 2165 2184GTCAGCCCCTGGTTACGGCG 5-10-5 70 34264 34283 786 547523 2167 2186TTGTCAGCCCCTGGTTACGG 5-10-5 69 34266 34285 787 547524 2169 2188CATTGTCAGCCCCTGGTTAC 5-10-5 58 34268 34287 788 546391 2170 2189GCATTGTCAGCCCCTGGTTA 5-10-5 54 34269 34288 789 547525 2174 2193CCTCGCATTGTCAGCCCCTG 5-10-5 78 34273 34292 790 546392 2176 2195GACCTCGCATTGTCAGCCCC 5-10-5 72 34275 34294 791 547526 2178 2197GCGACCTCGCATTGTCAGCC 5-10-5 59 34277 34296 792 547527 2185 2204CTCAGTTGCGACCTCGCATT 5-10-5 58 34284 34303 793 546393 2186 2205TCTCAGTTGCGACCTCGCAT 5-10-5 77 34285 34304 794 546394 2196 2215GTCATGGAGATCTCAGTTGC 5-10-5 71 34295 34314 795 547528 2200 2219CACAGTCATGGAGATCTCAG 5-10-5 78 34299 34318 796

TABLE 12 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 SEQ ISISStart Stop % Start Stop ID NO Site Site Sequence Motif inhibition SiteSite NO 531231 n/a n/a TATCACTGTACTAGTTTCCT 5-10-5 90 14744 14763 33414815 14834 14886 14905 14945 14964 15005 15024 15077 15096 15220 1523915292 15311 15351 15370 15411 15430 15483 15502 15555 15574 15613 1563215685 15704 15815 15834 15887 15906 15945 15964 546403 n/a n/aCCATGAACATCCTATCCGTG 5-10-5 83 3282 3301 797 546406 n/a n/aTGTCCTGTCAACATATTCCA 5-10-5 80 3299 3318 798 546409 n/a n/aGGGTTTCTGCCAACAGTTTC 5-10-5 77 3326 3345 799 546410 n/a n/aGACTTTGGGTTTCTGCCAAC 5-10-5 83 3332 3351 800 546411 n/a n/aATATTGACTTTGGGTTTCTG 5-10-5 56 3337 3356 801 546412 n/a n/aGGCTTCAATATTGACTTTGG 5-10-5 84 3344 3363 802 546416 n/a n/aCTGCAGGCAATATTTTGCTT 5-10-5 62 3364 3383 803 546418 n/a n/aATGTGGCACTGCAGGCAATA 5-10-5 72 3372 3391 804 546419 n/a n/aTTCTAATGTGGCACTGCAGG 5-10-5 65 3377 3396 805 546421 n/a n/aTCAAGCTGTTCTAATGTGGC 5-10-5 71 3385 3404 806 546422 n/a n/aACGGTCTTCAAGCTGTTCTA 5-10-5 72 3392 3411 807 546425 n/a n/aGGTCAATCTGACTAGTGAAT 5-10-5 69 2284 2303 808 546426 n/a n/aTCTCTGGTCAATCTGACTAG 5-10-5 49 2289 2308 809 546429 n/a n/aGCCCACCAACAATCTCTGGT 5-10-5 84 2301 2320 810 546432 n/a n/aGACCCCAACAGACAGCCCAC 5-10-5 62 2315 2334 811 546444 n/a n/aCCAGAATCATGCCTTGTGGG 5-10-5 61 4765 4784 812 546447 n/a n/aGTCACCATAGACCCAGAATC 5-10-5 68 4777 4796 813 546450 n/a n/aGTGGCCCTCTTAAGTCACCA 5-10-5 73 4790 4809 814 546453 n/a n/aCTCATTGTTGTGTGGCCCTC 5-10-5 82 4801 4820 815 546459 n/a n/aGTAGCCATACATCTGAGGAA 5-10-5 46 4830 4849 816 546461 n/a n/aATGTTTATTGTAGCCATACA 5-10-5 53 4839 4858 817 546492 n/a n/aCTCGCCTTTGTGACTCGATT 5-10-5 61 26263 26282 818 546493 n/a n/aCATACTCGCCTTTGTGACTC 5-10-5 35 26267 26286 819 546494 n/a n/aGCATACTCGCCTTTGTGACT 5-10-5 67 26268 26287 820 546495 n/a n/aTGCATACTCGCCTTTGTGAC 5-10-5 65 26269 26288 821 546395 2209 2228TTCACAACACACAGTCATGG 5-10-5 72 34308 34327 822 546397 2233 2252TTTTTTGATCTTTCACCATT 5-10-5 55 n/a n/a 823 546496 n/a n/aATGCATACTCGCCTTTGTGA 5-10-5 54 26270 26289 824 26301 26320 546497 n/an/a CATGCATACTCGCCTTTGTG 5-10-5 56 26271 26290 825 26302 26321 546498n/a n/a CCATGCATACTCGCCTTTGT 5-10-5 65 26272 26291 826 26303 26322547529 2203 2222 ACACACAGTCATGGAGATCT 5-10-5 49 34302 34321 827 5475302206 2225 ACAACACACAGTCATGGAGA 5-10-5 63 34305 34324 828 547531 22132232 TTATTTCACAACACACAGTC 5-10-5 69 34312 34331 829 546499 n/a n/aTCCATGCATACTCGCCTTTG 5-10-5 20 26273 26292 830 546500 n/a n/aTTCCATGCATACTCGCCTTT 5-10-5 46 26274 26293 831 546501 n/a n/aTTTCCATGCATACTCGCCTT 5-10-5 53 26275 26294 832 546502 n/a n/aGATTTTCCATGCATACTCGC 5-10-5 37 26278 26297 833 546503 n/a n/aGTGATGCGATTTTCCATGCA 5-10-5 53 26285 26304 834 546508 n/a n/aGCAGCAAGTGCTCCCCATGC 5-10-5 43 26317 26336 835 546511 n/a n/aGTGATGAAAGTACAGCAGCA 5-10-5 50 26331 26350 836 546683 n/a n/aTCCTATCCGTGTTCAGCTGT 5-10-5 69 3273 3292 837 546684 n/a n/aTACTCTCTACATACTCAGGA 5-10-5 71 3561 3580 838 546687 n/a n/aTGAGACCTCCAGACTACTGT 5-10-5 76 3847 3866 839 546690 n/a n/aCTCTGCTGGTTTTAGACCAC 5-10-5 44 4027 4046 840 546695 n/a n/aGGGACAATCTCCACCCCCGA 5-10-5 36 4225 4244 841 546698 n/a n/aTGCAGAGTGTCATCTGCGAA 5-10-5 59 4387 4406 842 546700 n/a n/aTGGTTCCCTAGCGGTCCAGA 5-10-5 78 4561 4580 843 546705 n/a n/aCCCCTGTAGTTGGCTGTGGT 5-10-5 66 5046 5065 844 546707 n/a n/aGCAAGTCAAAGAGTGTCCAC 5-10-5 73 5283 5302 845 546710 n/a n/aGAAGCCTGTTAGAGTTGGCC 5-10-5 73 5576 5595 846 546719 n/a n/aCCCCCATGTCCATGGACTTT 5-10-5 55 6329 6348 847 547532 n/a n/aCTGCCAACAGTTTCAACTTT 5-10-5 65 3320 3339 848 547533 n/a n/aTTTTGCTTGGCTTCAATATT 5-10-5 23 3352 3371 849 547534 n/a n/aATCTGACTAGTGAATGGCTT 5-10-5 72 2279 2298 850 547535 n/a n/aAGACAGCCCACCAACAATCT 5-10-5 28 2306 2325 851 547536 n/a n/aTGCATAGACCCCAACAGACA 5-10-5 48 2321 2340 852 547537 n/a n/aCCTGTGCATAGACCCCAACA 5-10-5 65 2325 2344 853 547538 n/a n/aCCAGCAGAAATCCTGTGCAT 5-10-5 77 2336 2355 854 547539 n/a n/aAGAACTCCAGCAGAAATCCT 5-10-5 43 2342 2361 855 547540 n/a n/aTTGTGTGGCCCTCTTAAGTC 5-10-5 44 4794 4813 856 547541 n/a n/aTATAGATGTTTATTGTAGCC 5-10-5 36 4844 4863 857 547542 n/a n/aATACTCGCCTTTGTGACTCG 5-10-5 35 26266 26285 858 547543 n/a n/aTTTTCCATGCATACTCGCCT 5-10-5 54 26276 26295 859 547544 n/a n/aTCGCCTTTGTGATGCGATTT 5-10-5 15 26293 26312 860 547545 n/a n/aATACTCGCCTTTGTGATGCG 5-10-5 43 26297 26316 861 547546 n/a n/aCATACTCGCCTTTGTGATGC 5-10-5 11 26298 26317 862 547547 n/a n/aGCATACTCGCCTTTGTGATG 5-10-5 42 26299 26318 863 547548 n/a n/aTGCATACTCGCCTTTGTGAT 5-10-5 61 26300 26319 864 547549 n/a n/aCCCATGCATACTCGCCTTTG 5-10-5 36 26304 26323 865 547550 n/a n/aCCCCATGCATACTCGCCTTT 5-10-5 53 26305 26324 866 547551 n/a n/aTCCCCATGCATACTCGCCTT 5-10-5 38 26306 26325 867 547552 n/a n/aCTCCCCATGCATACTCGCCT 5-10-5 53 26307 26326 868 547553 n/a n/aTGCTCCCCATGCATACTCGC 5-10-5 64 26309 26328 869 547554 n/a n/aGCTCTGATTGGGTCACCACA 5-10-5 50 5743 5762 870 547555 n/a n/aTGTCTCCTTCCACTTGCTCC 5-10-5 58 5923 5942 871 547556 n/a n/aGCCATTTTATCCCTGAGATT 5-10-5 55 6130 6149 872 547557 n/a n/aCTGTGCTGTATTTTGGAGCC 5-10-5 59 6413 6432 873

TABLE 13 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 SEQ ISISStart Stop % Start Stop ID NO Site Site Sequence Motif inhibition SiteSite NO 531231 n/a n/a TATCACTGTACTAGTTTCCT 5-10-5 85 14744 14763 33414815 14834 14886 14905 14945 14964 15005 15024 15077 15096 15220 1523915292 15311 15351 15370 15411 15430 15483 15502 15555 15574 15613 1563215685 15704 15815 15834 15887 15906 15945 15964 546732 n/a n/aGGATTTGGCCCTGAGCCCCA 5-10-5 14 6933 6952 874 546735 n/a n/aCAACCTGTCCATTCCCTGGG 5-10-5 46 7082 7101 875 546739 n/a n/aATTCGGTGTCTTTACTGGCT 5-10-5 89 7228 7247 876 546746 n/a n/aTCCTGTTGCCTGACATGCTA 5-10-5 65 7694 7713 877 546747 n/a n/aCTCCCACTGACTGACTACTC 5-10-5 64 7904 7923 878 546749 n/a n/aGCTGGTCCTTGAACCCCGTG 5-10-5 53 8259 8278 879 546753 n/a n/aCTGGCTCACTATAGGCCCCA 5-10-5 91 8655 8674 880 546756 n/a n/aATAAGCATCTCTCTGACCTA 5-10-5 47 9105 9124 881 546763 n/a n/aGCTTCCCCAATACTTGCTGG 5-10-5 84 9695 9714 882 546765 n/a n/aGTGTCCAGAATACTGCCCCA 5-10-5 82 10053 10072 883 546770 n/a n/aGTGGACGACTGCCCTGTGCC 5-10-5 74 10435 10454 884 546773 n/a n/aTCTCTAGCATCCTAGTCCTC 5-10-5 67 10586 10605 885 546780 n/a n/aATACTGGCTAAGTCAGGCCC 5-10-5 83 10982 11001 886 546784 n/a n/aGGCAGGGAGGTGGATTATTC 5-10-5 58 11440 11459 887 546789 n/a n/aGCTTCTCTATCTCCCAGTGT 5-10-5 79 12228 12247 888 546791 n/a n/aGATGCATGCAGCAATACAGG 5-10-5 52 12385 12404 889 546795 n/a n/aGTCTCGATGGCAAGCTGTAC 5-10-5 72 12650 12669 890 546796 n/a n/aGTACTCACCGGTACTCTGCC 5-10-5 82 12804 12823 891 546799 n/a n/aATGAAGGGCGAGGCGCAGTG 5-10-5 5 13258 13277 892 546803 n/a n/aCCCCATACATCTATGCAAAT 5-10-5 40 13551 13570 893 546804 n/a n/aACATGACTCCAGTGATGGAT 5-10-5 57 13632 13651 894 546808 n/a n/aAAAATGACACCAAAATTCGC 5-10-5 0 13841 13860 895 546811 n/a n/aTGGACATCCTTCCCCTCGCA 5-10-5 49 13967 13986 896 546817 n/a n/aGCTCTGAGCCTTCCGCCTCT 5-10-5 77 14472 14491 897 546822 n/a n/aACTAGTTTCCTATAACTGCT 5-10-5 32 14735 14754 898 546823 n/a n/aTACTAGTTTCCTATAACTGC 5-10-5 44 14736 14755 899 546824 n/a n/aGTACTAGTTTCCTATAACTG 5-10-5 79 14737 14756 900 546825 n/a n/aGTATCACTGTACTAGTTTCC 5-10-5 96 14745 14764 901 14816 14835 14887 1490614946 14965 15006 15025 15078 15097 15221 15240 15293 15312 15352 1537115412 15431 15484 15503 15556 15575 15614 15633 15686 15705 15816 1583515888 15907 15946 15965 546826 n/a n/a AGTATCACTGTACTAGTTTC 5-10-5 9014746 14765 902 14817 14836 14888 14907 14947 14966 15007 15026 1507915098 15222 15241 15294 15313 15353 15372 15413 15432 15485 15504 1555715576 15615 15634 15687 15706 15817 15836 15889 15908 15947 15966 546827n/a n/a CAGTATCACTGTACTAGTTT 5-10-5 98 14747 14766 903 14818 14837 1488914908 14948 14967 15008 15027 15080 15099 15152 15171 15223 15242 1529515314 15354 15373 15414 15433 15486 15505 15558 15577 15616 15635 1568815707 15818 15837 15890 15909 15948 15967 546828 n/a n/aACAGTATCACTGTACTAGTT 5-10-5 95 14748 14767 904 14819 14838 14890 1490914949 14968 15009 15028 15081 15100 15153 15172 15224 15243 15296 1531515355 15374 15415 15434 15487 15506 15559 15578 15617 15636 15689 1570815819 15838 15891 15910 15949 15968 546829 n/a n/a AACAGTATCACTGTACTAGT5-10-5 94 14749 14768 905 14820 14839 14891 14910 14950 14969 1501015029 15082 15101 15154 15173 15225 15244 15297 15316 15356 15375 1541615435 15488 15507 15560 15579 15618 15637 15690 15709 15820 15839 1589215911 15950 15969 546830 n/a n/a TAACAGTATCACTGTACTAG 5-10-5 78 1475014769 906 14821 14840 14892 14911 14951 14970 15011 15030 15083 1510215155 15174 15226 15245 15298 15317 15357 15376 15417 15436 15489 1550815561 15580 15619 15638 15691 15710 15821 15840 15893 15912 15951 15970546831 n/a n/a TCTAACAGTATCACTGTACT 5-10-5 79 14752 14771 907 1482314842 14894 14913 15013 15032 15085 15104 15228 15247 15300 15319 1541915438 15491 15510 15621 15640 15823 15842 15953 15972 546832 n/a n/aCTCTAACAGTATCACTGTAC 5-10-5 88 14753 14772 908 14824 14843 14895 1491415014 15033 15086 15105 15229 15248 15301 15320 15420 15439 15492 1551115622 15641 15824 15843 15954 15973 546833 n/a n/a ACTCTAACAGTATCACTGTA5-10-5 90 14754 14773 909 14825 14844 14896 14915 15015 15034 1508715106 15230 15249 15302 15321 15421 15440 15493 15512 15623 15642 1582515844 15955 15974 546834 n/a n/a AACTCTAACAGTATCACTGT 5-10-5 86 1475514774 910 14826 14845 14897 14916 15016 15035 15088 15107 15231 1525015303 15322 15422 15441 15494 15513 15624 15643 15826 15845 15956 15975546835 n/a n/a TAACTCTAACAGTATCACTG 5-10-5 86 14756 14775 911 1482714846 14898 14917 15017 15036 15089 15108 15232 15251 15304 15323 1542315442 15495 15514 15625 15644 15827 15846 15957 15976 546836 n/a n/aATAACTCTAACAGTATCACT 5-10-5 30 14757 14776 912 14828 14847 14899 1491815018 15037 15090 15109 15233 15252 15305 15324 15424 15443 15496 1551515626 15645 15828 15847 15958 15977 546837 n/a n/a TATAACTCTAACAGTATCAC5-10-5 0 14758 14777 913 14829 14848 14900 14919 15019 15038 15091 1511015234 15253 15306 15325 15425 15444 15497 15516 15627 15646 15829 1584815959 15978 546838 n/a n/a CTATAACTCTAACAGTATCA 5-10-5 43 14759 14778914 14830 14849 14901 14920 15020 15039 15092 15111 15235 15254 1530715326 15426 15445 15498 15517 15628 15647 15830 15849 15960 15979 546839n/a n/a CCTATAACTCTAACAGTATC 5-10-5 47 14760 14779 915 14831 14850 1490214921 15021 15040 15093 15112 15236 15255 15308 15327 15427 15446 1549915518 15629 15648 15831 15850 15961 15980 546840 n/a n/aCTGTCCTATAACTCTAACAG 5-10-5 53 14764 14783 916 14835 14854 546841 n/an/a CACTGTCCTATAACTCTAAC 5-10-5 38 14766 14785 917 14837 14856 546842n/a n/a TCACTGTCCTATAACTCTAA 5-10-5 54 14767 14786 918 14838 14857546843 n/a n/a TATCACTGTCCTATAACTCT 5-10-5 52 14769 14788 919 1484014859 546844 n/a n/a GTCCTATATCACTGTCCTAT 5-10-5 75 14775 14794 92014846 14865 15180 15199 15716 15735 546845 n/a n/a TGTCCTATATCACTGTCCTA5-10-5 75 14776 14795 921 14847 14866 15181 15200 15717 15736 546846 n/an/a CTGTCCTATATCACTGTCCT 5-10-5 95 14777 14796 922 14848 14867 1518215201 15718 15737 546847 n/a n/a ACTGTCCTATATCACTGTCC 5-10-5 88 1477814797 923 14849 14868 15183 15202 15719 15738 546848 n/a n/aTCACTGTCCTATATCACTGT 5-10-5 86 14780 14799 924 14851 14870 14976 1499515185 15204 15257 15276 15382 15401 15520 15539 15650 15669 15721 1574015852 15871 15982 16001 547558 n/a n/a CCCCCAGTTCCCATGCAAGG 5-10-5 526640 6659 925 547559 n/a n/a GAGCACAGATCTCTTCAAGT 5-10-5 69 6822 6841926 547560 n/a n/a GACGGTCACCCAGCCCTGAC 5-10-5 42 7459 7478 927 547561n/a n/a AAGGGAAATTAGAGGCAGGC 5-10-5 57 7583 7602 928 547562 n/a n/aCTTTCTTGAGACAATCCCTT 5-10-5 59 8463 8482 929 547563 n/a n/aGTGGGATCAGAGAATGACTA 5-10-5 48 9267 9286 930 547564 n/a n/aCCCTCTGTCTTAGATGTCCA 5-10-5 94 9390 9409 931 547565 n/a n/aCTTATCAGTCCCAGTCATGT 5-10-5 63 10698 10717 932 547566 n/a n/aAAGAGTTGGGATGCGACTCT 5-10-5 76 11335 11354 933 547567 n/a n/aTCCACTCCTAAGAAGTATGG 5-10-5 60 11546 11565 934 547568 n/a n/aGCACCCTTTTCATTGAGATT 5-10-5 70 12070 12089 935 547569 n/a n/aACTACCATTTGGGTTGGTAG 5-10-5 9 12571 12590 936 547570 n/a n/aAAGCCCTGTTTGGTTTTTAG 5-10-5 18 12900 12919 937 547571 n/a n/aAAATGACACCAAAATTGAGT 5-10-5 14 13744 13763 938 547572 n/a n/aAAATGACACCAAAATTCGCT 5-10-5 40 13840 13859 939 547573 n/a n/aTAAGCAAGGCCTATGTGTGG 5-10-5 2 13880 13899 940 547574 n/a n/aACACGCACAGGTCCCAGGGC 5-10-5 51 14314 14333 941 547575 n/a n/aGGGAAACTCTTTCCTCGCCC 5-10-5 89 14583 14602 942 547576 n/a n/aCTAGTTTCCTATAACTGCTG 5-10-5 29 14734 14753 943 547577 n/a n/aCTAACAGTATCACTGTACTA 5-10-5 79 14751 14770 944 14822 14841 14893 1491215012 15031 15084 15103 15227 15246 15299 15318 15418 15437 15490 1550915620 15639 15822 15841 15952 15971 547578 n/a n/a GTCCTATAACTCTAACAGTA5-10-5 30 14762 14781 945 14833 14852 547579 n/a n/aTGTCCTATAACTCTAACAGT 5-10-5 0 14763 14782 946 14834 14853 547580 n/a n/aATCACTGTCCTATAACTCTA 5-10-5 61 14768 14787 947 14839 14858 547581 n/an/a ATATCACTGTCCTATAACTC 5-10-5 60 14770 14789 948 14841 14860 547582n/a n/a TATATCACTGTCCTATAACT 5-10-5 22 14771 14790 949 14842 14861 1517615195 15712 15731 16160 16179 547583 n/a n/a CACTGTCCTATATCACTGTC 5-10-580 14779 14798 950 14850 14869 15184 15203 15720 15739

TABLE 14 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 SEQ ISISStart Stop % Start Stop ID NO Site Site Sequence Motif inhibition SiteSite NO 531231 n/a n/a TATCACTGTACTAGTTTCCT 5-10-5 85 14744 14763 33414815 14834 14886 14905 14945 14964 15005 15024 15077 15096 15220 1523915292 15311 15351 15370 15411 15430 15483 15502 15555 15574 15613 1563215685 15704 15815 15834 15887 15906 15945 15964 546849 n/a n/aATCACTGTCCTATATCACTG 5-10-5 93 14781 14800 951 14852 14871 14977 1499615186 15205 15258 15277 15383 15402 15521 15540 15651 15670 15722 1574115853 15872 15983 16002 546850 n/a n/a TATCACTGTCCTATATCACT 5-10-5 8014782 14801 952 14853 14872 14978 14997 15116 15135 15187 15206 1525915278 15384 15403 15522 15541 15652 15671 15723 15742 15854 15873 1598416003 546851 n/a n/a AGTATCACTGTCCTATATCA 5-10-5 81 14784 14803 95314980 14999 15118 15137 15386 15405 15524 15543 15986 16005 546852 n/an/a CAGTATCACTGTCCTATATC 5-10-5 94 14785 14804 954 14981 15000 1511915138 15387 15406 15525 15544 15987 16006 546853 n/a n/aACAGTATCACTGTCCTATAT 5-10-5 86 14786 14805 955 14982 15001 15120 1513915388 15407 15526 15545 15988 16007 546854 n/a n/a TAACAGTATCACTGTCCTAT5-10-5 90 14788 14807 956 14984 15003 15050 15069 15122 15141 1539015409 15456 15475 15528 15547 15990 16009 546855 n/a n/aATAACAGTATCACTGTCCTA 5-10-5 87 14789 14808 957 14985 15004 15051 1507015123 15142 15391 15410 15457 15476 15529 15548 15991 16010 546856 n/an/a AACTATAACAGTATCACTGT 5-10-5 54 14793 14812 958 15055 15074 1512715146 15160 15179 15461 15480 15533 15552 15566 15585 15696 15715 1589815917 15995 16014 546857 n/a n/a TATAACTATAACAGTATCAC 5-10-5 7 1479614815 959 15058 15077 15130 15149 15163 15182 15464 15483 15536 1555515569 15588 15699 15718 15770 15789 15998 16017 546858 n/a n/aCTATAACTATAACAGTATCA 5-10-5 21 14797 14816 960 15059 15078 15131 1515015164 15183 15465 15484 15537 15556 15570 15589 15700 15719 15771 1579015999 16018 546859 n/a n/a TTTCCTATAACTATAACAGT 5-10-5 7 14801 14820 96115063 15082 15469 15488 15541 15560 546860 n/a n/a CTAGTTTCCTATAACTATAA5-10-5 36 14805 14824 962 14876 14895 14935 14954 15067 15086 1521015229 15282 15301 15341 15360 15473 15492 15545 15564 15603 15622 1567515694 15746 15765 15805 15824 15877 15896 15935 15954 546861 n/a n/aTAACAATATCACTGTCCTAT 5-10-5 68 14859 14878 963 15193 15212 15265 1528415586 15605 15658 15677 15729 15748 15860 15879 16086 16105 16183 1620216234 16253 546862 n/a n/a AACTATAACAATATCACTGT 5-10-5 0 14864 14883 96414923 14942 15198 15217 15270 15289 15329 15348 15591 15610 15663 1568215734 15753 15793 15812 15865 15884 15923 15942 16066 16085 16091 1611016144 16163 16239 16258 546863 n/a n/a TAACTATAACAATATCACTG 5-10-5 2114865 14884 965 14924 14943 15199 15218 15271 15290 15330 15349 1559215611 15664 15683 15735 15754 15794 15813 15866 15885 15924 15943 1606716086 16092 16111 16145 16164 16240 16259 546864 n/a n/aATAACTATAACAATATCACT 5-10-5 0 14866 14885 966 14925 14944 15200 1521915272 15291 15331 15350 15593 15612 15665 15684 15736 15755 15795 1581415867 15886 15925 15944 16068 16087 16093 16112 16146 16165 16241 16260546865 n/a n/a TATAACTATAACAATATCAC 5-10-5 0 14867 14886 967 14926 1494515201 15220 15273 15292 15332 15351 15594 15613 15666 15685 15737 1575615796 15815 15868 15887 15926 15945 16069 16088 16094 16113 16147 1616616242 16261 546866 n/a n/a GTTTCCTATAACTATAACAA 5-10-5 35 14873 14892968 14932 14951 15207 15226 15279 15298 15338 15357 15600 15619 1567215691 15743 15762 15802 15821 15874 15893 15932 15951 546867 n/a n/aACCTATAACTCTAACAGTAT 5-10-5 40 14903 14922 969 15022 15041 15094 1511315237 15256 15309 15328 15428 15447 15500 15519 15630 15649 15832 1585115962 15981 546868 n/a n/a TACCTATAACTCTAACAGTA 5-10-5 51 14904 14923970 15023 15042 15095 15114 15238 15257 15310 15329 15429 15448 1550115520 15631 15650 15833 15852 15963 15982 546869 n/a n/aTGTACCTATAACTCTAACAG 5-10-5 53 14906 14925 971 15025 15044 15240 1525915312 15331 15431 15450 15503 15522 15633 15652 15835 15854 15965 15984546870 n/a n/a CTGTACCTATAACTCTAACA 5-10-5 87 14907 14926 972 1502615045 15241 15260 15313 15332 15432 15451 15504 15523 15634 15653 1583615855 15966 15985 546871 n/a n/a ACTGTACCTATAACTCTAAC 5-10-5 73 1490814927 973 15027 15046 15242 15261 15314 15333 15433 15452 15505 1552415635 15654 15837 15856 15967 15986 546872 n/a n/a CACTGTACCTATAACTCTAA5-10-5 87 14909 14928 974 15028 15047 15243 15262 15315 15334 1543415453 15506 15525 15636 15655 15838 15857 15968 15987 546873 n/a n/aCAATATCACTGTACCTATAA 5-10-5 34 14915 14934 975 15321 15340 15785 15804546874 n/a n/a ATAACAATATCACTGTACCT 5-10-5 68 14919 14938 976 1532515344 15789 15808 16062 16081 16140 16159 546875 n/a n/aACTATAACAATATCACTGTA 5-10-5 33 14922 14941 977 15328 15347 15792 1581116065 16084 16143 16162 546876 n/a n/a GTCCTATATCACTGTACCTG 5-10-5 8714971 14990 978 546877 n/a n/a CACTGTCCTATATCACTGTA 5-10-5 88 1497514994 979 15256 15275 15381 15400 15519 15538 15649 15668 15851 1587015981 16000 546878 n/a n/a CCTATAACAGTATCACTGTC 5-10-5 81 14988 15007980 15394 15413 546879 n/a n/a TTTCCTATAACAGTATCACT 5-10-5 42 1499115010 981 15397 15416 546880 n/a n/a GTTTCCTATAACAGTATCAC 5-10-5 4114992 15011 982 15398 15417 546881 n/a n/a AGTTTCCTATAACAGTATCA 5-10-549 14993 15012 983 15399 15418 546882 n/a n/a TAGTTTCCTATAACAGTATC5-10-5 24 14994 15013 984 15400 15419 546883 n/a n/aCTAGTTTCCTATAACAGTAT 5-10-5 19 14995 15014 985 15401 15420 546884 n/an/a ACTAGTTTCCTATAACAGTA 5-10-5 6 14996 15015 986 15402 15421 547584 n/an/a GTATCACTGTCCTATATCAC 5-10-5 85 14783 14802 987 14979 14998 1511715136 15385 15404 15523 15542 15985 16004 547585 n/a n/aAACAGTATCACTGTCCTATA 5-10-5 85 14787 14806 988 14983 15002 15121 1514015389 15408 15527 15546 15989 16008 547586 n/a n/a TATAACAGTATCACTGTCCT5-10-5 82 14790 14809 989 14986 15005 15052 15071 15124 15143 1539215411 15458 15477 15530 15549 15992 16011 547587 n/a n/aCTATAACAGTATCACTGTCC 5-10-5 96 14791 14810 990 14987 15006 15053 1507215125 15144 15393 15412 15459 15478 15531 15550 15993 16012 547588 n/an/a ACTATAACAGTATCACTGTC 5-10-5 83 14792 14811 991 15054 15073 1512615145 15460 15479 15532 15551 15994 16013 547589 n/a n/aTAACTATAACAGTATCACTG 5-10-5 36 14794 14813 992 15056 15075 15128 1514715161 15180 15462 15481 15534 15553 15567 15586 15697 15716 15996 16015547590 n/a n/a ATAACTATAACAGTATCACT 5-10-5 0 14795 14814 993 15057 1507615129 15148 15162 15181 15463 15482 15535 15554 15568 15587 15698 1571715997 16016 547591 n/a n/a CCTATAACTATAACAGTATC 5-10-5 23 14798 14817994 15060 15079 15165 15184 15466 15485 15538 15557 15571 15590 1570115720 15772 15791 16000 16019 547592 n/a n/a TCCTATAACTATAACAGTAT 5-10-527 14799 14818 995 15061 15080 15166 15185 15467 15486 15539 15558 1557215591 15702 15721 16001 16020 547593 n/a n/a TTCCTATAACTATAACAGTA 5-10-529 14800 14819 996 15062 15081 15468 15487 15540 15559 547594 n/a n/aGTTTCCTATAACTATAACAG 5-10-5 19 14802 14821 997 15064 15083 15470 1548915542 15561 547595 n/a n/a ACTAGTTTCCTATAACTATA 5-10-5 21 14806 14825998 14877 14896 14936 14955 15068 15087 15211 15230 15283 15302 1534215361 15474 15493 15546 15565 15604 15623 15676 15695 15747 15766 1580615825 15878 15897 15936 15955 547596 n/a n/a TACTAGTTTCCTATAACTAT 5-10-514 14807 14826 999 14878 14897 14937 14956 15069 15088 15212 15231 1528415303 15343 15362 15475 15494 15547 15566 15605 15624 15677 15696 1574815767 15807 15826 15879 15898 15937 15956 547597 n/a n/aCAATATCACTGTCCTATATC 5-10-5 29 14856 14875 1000 15190 15209 15262 1528115655 15674 15726 15745 15857 15876 547598 n/a n/a ACTATAACAATATCACTGTC5-10-5 59 14863 14882 1001 15197 15216 15269 15288 15590 15609 1566215681 15733 15752 15864 15883 15922 15941 16090 16109 16238 16257 547599n/a n/a TTCCTATAACTATAACAATA 5-10-5 4 14871 14890 1002 14930 14949 1520515224 15277 15296 15336 15355 15598 15617 15670 15689 15741 15760 1580015819 15872 15891 15930 15949 547600 n/a n/a TTTCCTATAACTATAACAAT 5-10-526 14872 14891 1003 14931 14950 15206 15225 15278 15297 15337 1535615599 15618 15671 15690 15742 15761 15801 15820 15873 15892 15931 15950547601 n/a n/a GTACCTATAACTCTAACAGT 5-10-5 75 14905 14924 1004 1502415043 15239 15258 15311 15330 15430 15449 15502 15521 15632 15651 1583415853 15964 15983 547602 n/a n/a TCACTGTACCTATAACTCTA 5-10-5 93 1491014929 1005 15029 15048 15244 15263 15316 15335 15435 15454 15507 1552615637 15656 15839 15858 15969 15988 547603 n/a n/a TATCACTGTACCTATAACTC5-10-5 41 14912 14931 1006 15246 15265 15318 15337 15509 15528 1563915658 15841 15860 15971 15990 547604 n/a n/a ATATCACTGTACCTATAACT 5-10-50 14913 14932 1007 15247 15266 15319 15338 15510 15529 15640 15659 1578315802 15842 15861 15972 15991 547605 n/a n/a ACAATATCACTGTACCTATA 5-10-543 14916 14935 1008 15322 15341 15786 15805 16137 16156 547606 n/a n/aAACAATATCACTGTACCTAT 5-10-5 43 14917 14936 1009 15323 15342 15787 1580616138 16157 547607 n/a n/a TAACAATATCACTGTACCTA 5-10-5 49 14918 149371010 15324 15343 15788 15807 16139 16158 547608 n/a n/aTATAACAATATCACTGTACC 5-10-5 35 14920 14939 1011 15326 15345 15790 1580916063 16082 16141 16160 547609 n/a n/a CTATAACAATATCACTGTAC 5-10-5 2314921 14940 1012 15327 15346 15791 15810 16064 16083 16142 16161 547610n/a n/a TGTAACAGTATCACTGTACT 5-10-5 45 14953 14972 1013 547611 n/a n/aCTGTAACAGTATCACTGTAC 5-10-5 71 14954 14973 1014 547612 n/a n/aCCTGTAACAGTATCACTGTA 5-10-5 68 14955 14974 1015 547613 n/a n/aCTATATCACTGTACCTGTAA 5-10-5 39 14968 14987 1016 547614 n/a n/aCCTATATCACTGTACCTGTA 5-10-5 81 14969 14988 1017 547615 n/a n/aTCCTATATCACTGTACCTGT 5-10-5 84 14970 14989 1018 547616 n/a n/aTGTCCTATATCACTGTACCT 5-10-5 86 14972 14991 1019 15253 15272 15378 1539715516 15535 15646 15665 15848 15867 15978 15997 547617 n/a n/aCTGTCCTATATCACTGTACC 5-10-5 91 14973 14992 1020 15254 15273 15379 1539815517 15536 15647 15666 15849 15868 15979 15998 547618 n/a n/aACTGTCCTATATCACTGTAC 5-10-5 87 14974 14993 1021 15255 15274 15380 1539915518 15537 15648 15667 15850 15869 15980 15999 547619 n/a n/aTCCTATAACAGTATCACTGT 5-10-5 70 14989 15008 1022 15395 15414 547620 n/an/a TTCCTATAACAGTATCACTG 5-10-5 65 14990 15009 1023 15396 15415 547621n/a n/a TACTAGTTTCCTATAACAGT 5-10-5 12 14997 15016 1024 15403 15422547622 n/a n/a GTCACTGTACCTATAACTCT 5-10-5 88 15030 15049 1025 1543615455 547623 n/a n/a TGTCACTGTACCTATAACTC 5-10-5 81 15031 15050 102615437 15456 547624 n/a n/a ATGTCACTGTACCTATAACT 5-10-5 64 15032 150511027 15438 15457

TABLE 15 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 ISISStart Stop % Start Stop SEQ ID NO Site Site Sequence inhibition MotifSite Site NO 531231 n/a n/a TATCACTGTACTAGTTTCCT 93 5-10-5 14744 14763334 14815 14834 14886 14905 14945 14964 15005 15024 15077 15096 1522015239 15292 15311 15351 15370 15411 15430 15483 15502 15555 15574 1561315632 15685 15704 15815 15834 15887 15906 15945 15964 546885 n/a n/aTATGTCACTGTACCTATAAC 46 5-10-5 15033 15052 1028 15439 15458 546886 n/an/a CTATGTCACTGTACCTATAA 80 5-10-5 15034 15053 1029 15440 15459 546887n/a n/a CCTATGTCACTGTACCTATA 82 5-10-5 15035 15054 1030 15441 15460546888 n/a n/a TCCTATGTCACTGTACCTAT 78 5-10-5 15036 15055 1031 1544215461 546889 n/a n/a GTCCTATGTCACTGTACCTA 93 5-10-5 15037 15056 103215443 15462 546890 n/a n/a TGTCCTATGTCACTGTACCT 78 5-10-5 15038 150571033 15444 15463 546891 n/a n/a CTGTCCTATGTCACTGTACC 81 5-10-5 1503915058 1034 15445 15464 546892 n/a n/a ACTGTCCTATGTCACTGTAC 82 5-10-515040 15059 1035 15446 15465 546893 n/a n/a CACTGTCCTATGTCACTGTA 705-10-5 15041 15060 1036 15447 15466 546894 n/a n/a TCACTGTCCTATGTCACTGT91 5-10-5 15042 15061 1037 15448 15467 546895 n/a n/aTATCACTGTCCTATGTCACT 77 5-10-5 15044 15063 1038 15450 15469 546896 n/an/a GTATCACTGTCCTATGTCAC 75 5-10-5 15045 15064 1039 15451 15470 546897n/a n/a AGTATCACTGTCCTATGTCA 90 5-10-5 15046 15065 1040 15452 15471546898 n/a n/a AACAGTATCACTGTCCTATG 91 5-10-5 15049 15068 1041 1545515474 546899 n/a n/a CTACCTATAACTCTAACAGT 27 5-10-5 15096 15115 1042546901 n/a n/a ACTGTCCTATAACTATAACA 56 5-10-5 15170 15189 1043 1557615595 15706 15725 16005 16024 16076 16095 16101 16120 16154 16173 546902n/a n/a CACTGTCCTATAACTATAAC 71 5-10-5 15171 15190 1044 15577 1559615707 15726 16006 16025 16077 16096 16102 16121 16155 16174 546903 n/an/a CCTATATCACTGTACCTATA 91 5-10-5 15250 15269 1045 15375 15394 1551315532 15643 15662 15845 15864 15975 15994 546904 n/a n/aTCCTATATCACTGTACCTAT 80 5-10-5 15251 15270 1046 15376 15395 15514 1553315644 15663 15846 15865 15976 15995 546905 n/a n/a TACCTATAACAGTATCACTG65 5-10-5 15363 15382 1047 546907 n/a n/a ATAACTATAACAGTATCACC 37 5-10-515769 15788 1048 546908 n/a n/a TCACTGTACCTATAACTATA 77 5-10-5 1578015799 1049 16252 16271 546909 n/a n/a AACAATATCACTGTACCTTT 44 5-10-516060 16079 1050 546910 n/a n/a TAACAATATCACTGTACCTT 82 5-10-5 1606116080 1051 546911 n/a n/a GTCCTATAACTATAACAATA 52 5-10-5 16073 160921052 16098 16117 16151 16170 547625 n/a n/a CAGTATCACTGTCCTATGTC 795-10-5 15047 15066 1053 15453 15472 547626 n/a n/a ACAGTATCACTGTCCTATGT91 5-10-5 15048 15067 1054 15454 15473 547627 n/a n/aTCTACCTATAACTCTAACAG 71 5-10-5 15097 15116 1055 547628 n/a n/aCTCTACCTATAACTCTAACA 34 5-10-5 15098 15117 1056 547629 n/a n/aACTCTACCTATAACTCTAAC 0 5-10-5 15099 15118 1057 547630 n/a n/aACTGTCCTATATCACTCTAC 76 5-10-5 15112 15131 1058 547631 n/a n/aCACTGTCCTATATCACTCTA 85 5-10-5 15113 15132 1059 547632 n/a n/aTCACTGTCCTATATCACTCT 87 5-10-5 15114 15133 1060 547633 n/a n/aATCACTGTCCTATATCACTC 87 5-10-5 15115 15134 1061 547634 n/a n/aATCACTGTACTAGTTTTCTA 72 5-10-5 15148 15167 1062 547635 n/a n/aTATCACTGTACTAGTTTTCT 53 5-10-5 15149 15168 1063 547636 n/a n/aGTATCACTGTACTAGTTTTC 86 5-10-5 15150 15169 1064 547637 n/a n/aAGTATCACTGTACTAGTTTT 88 5-10-5 15151 15170 1065 547638 n/a n/aATAACAGTATCACTGTACTA 87 5-10-5 15156 15175 1066 15358 15377 15562 1558115692 15711 15894 15913 547639 n/a n/a GTCCTATAACTATAACAGTA 72 5-10-515167 15186 1067 15573 15592 15703 15722 16002 16021 547640 n/a n/aTGTCCTATAACTATAACAGT 13 5-10-5 15168 15187 1068 15574 15593 15704 1572316003 16022 547641 n/a n/a CTGTCCTATAACTATAACAG 43 5-10-5 15169 151881069 15575 15594 15705 15724 16004 16023 547642 n/a n/aTCACTGTCCTATAACTATAA 72 5-10-5 15172 15191 1070 15578 15597 15708 1572716007 16026 16078 16097 16103 16122 16156 16175 547643 n/a n/aATCACTGTCCTATAACTATA 72 5-10-5 15173 15192 1071 15579 15598 15709 1572816008 16027 16079 16098 16104 16123 16157 16176 16176 16195 547644 n/an/a TATCACTGTCCTATAACTAT 51 5-10-5 15174 15193 1072 15580 15599 1571015729 16009 16028 16080 16099 16158 16177 16177 16196 16228 16247 547645n/a n/a ATATCACTGTCCTATAACTA 60 5-10-5 15175 15194 1073 15581 1560015711 15730 16010 16029 16081 16100 16159 16178 16178 16197 16229 16248547646 n/a n/a CTATATCACTGTACCTATAA 23 5-10-5 15249 15268 1074 1537415393 15512 15531 15642 15661 15844 15863 15974 15993 547647 n/a n/aGTCCTATATCACTGTACCTA 92 5-10-5 15252 15271 1075 15377 15396 15515 1553415645 15664 15847 15866 15977 15996 547648 n/a n/a CCTATAACAGTATCACTGTA83 5-10-5 15361 15380 1076 547649 n/a n/a ACCTATAACAGTATCACTGT 73 5-10-515362 15381 1077 547650 n/a n/a GTACCTATAACAGTATCACT 32 5-10-5 1536415383 1078 547651 n/a n/a TGTACCTATAACAGTATCAC 48 5-10-5 15365 153841079 547652 n/a n/a TCACTGTACCTATAACAGTA 59 5-10-5 15369 15388 1080547653 n/a n/a ATCACTGTACCTATAACAGT 57 5-10-5 15370 15389 1081 547654n/a n/a TATCACTGTACCTATAACAG 53 5-10-5 15371 15390 1082 547655 n/a n/aAATATCACTGTCCTATAACT 37 5-10-5 15582 15601 1083 16011 16030 16082 1610116179 16198 16230 16249 547656 n/a n/a CAATATCACTGTCCTATAAC 42 5-10-515583 15602 1084 16083 16102 16180 16199 16231 16250 547657 n/a n/aACAATATCACTGTCCTATAA 43 5-10-5 15584 15603 1085 16084 16103 16181 1620016232 16251 547658 n/a n/a CGTACTAGTTTCCTATAACT 68 5-10-5 15750 157691086 547659 n/a n/a ACTATAACAGTATCACCGTA 80 5-10-5 15766 15785 1087547660 n/a n/a AACTATAACAGTATCACCGT 68 5-10-5 15767 15786 1088 547661n/a n/a TAACTATAACAGTATCACCG 80 5-10-5 15768 15787 1089 547662 n/a n/aACCTATAACTATAACAGTAT 0 5-10-5 15773 15792 1090 547663 n/a n/aTACCTATAACTATAACAGTA 10 5-10-5 15774 15793 1091 547664 n/a n/aGTACCTATAACTATAACAGT 2 5-10-5 15775 15794 1092 547665 n/a n/aTGTACCTATAACTATAACAG 10 5-10-5 15776 15795 1093 547666 n/a n/aATCACTGTACCTATAACTAT 71 5-10-5 15781 15800 1094 16253 16272 547667 n/an/a TATCACTGTACCTATAACTA 55 5-10-5 15782 15801 1095 547668 n/a n/aCAACTATAACAGTATCACTG 44 5-10-5 15899 15918 1096 547669 n/a n/aACAACTATAACAGTATCACT 0 5-10-5 15900 15919 1097 547670 n/a n/aTACAACTATAACAGTATCAC 0 5-10-5 15901 15920 1098 547671 n/a n/aCTACAACTATAACAGTATCA 0 5-10-5 15902 15921 1099 547672 n/a n/aCAATATCACTGTCCTACAAC 36 5-10-5 15915 15934 1100 547673 n/a n/aGAATATCACTGTCCTATAAC 21 5-10-5 16012 16031 1101 547674 n/a n/aACAATATCACTGTACCTTTA 53 5-10-5 16059 16078 1102 547675 n/a n/aTGTCCTATAACTATAACAAT 10 5-10-5 16074 16093 1103 16099 16118 16152 16171547676 n/a n/a CTGTCCTATAACTATAACAA 41 5-10-5 16075 16094 1104 1610016119 16153 16172

TABLE 16 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 SEQ ISISStart Stop % Start Stop ID NO Site Site Sequence Motif inhibition SiteSite NO 531231 n/a n/a TATCACTGTACTAGTTTCCT 5-10-5 93 14744 14763 33414815 14834 14886 14905 14945 14964 15005 15024 15077 15096 15220 1523915292 15311 15351 15370 15411 15430 15483 15502 15555 15574 15613 1563215685 15704 15815 15834 15887 15906 15945 15964 546529 n/a n/aGCACCTGGCAGAACAGTACC 5-10-5 65 26419 26438 1105 546578 n/a n/aGACAGTGGGCCAGAGCCTTG 5-10-5 73 26686 26705 1106 546912 n/a n/aACATCACTGTCCTATAACTA 5-10-5 26 16106 16125 1107 546913 n/a n/aGTACCTATATCACTGTAACT 5-10-5 38 16126 16145 1108 546914 n/a n/aATATCACTGTACCTATATCA 5-10-5 52 16134 16153 1109 546915 n/a n/aTCACTGTCCTATAACTATAT 5-10-5 39 16175 16194 1110 546916 n/a n/aCGTCACTGTACCTATAACTG 5-10-5 92 16203 16222 1111 546917 n/a n/aATCACTGTCCTATAACTATT 5-10-5 63 16227 16246 1112 546918 n/a n/aAACATCACTGTACCTATAAC 5-10-5 14 16256 16275 1113 546926 n/a n/aGCCATCCAGGGTGCTCTCCC 5-10-5 81 16839 16858 1114 546931 n/a n/aGCCCCCGGAGCACCTTCACT 5-10-5 58 17205 17224 1115 546935 n/a n/aCGTGGTTAGCCTGACATCTC 5-10-5 86 17412 17431 1116 546939 n/a n/aGCCATCTGGTTAGCCTCCGA 5-10-5 89 17664 17683 1117 546942 n/a n/aTACACTGAACCCCCTTAGGC 5-10-5 56 18570 18589 1118 546943 n/a n/aCAGTTTGGCCTTTCCATCTC 5-10-5 54 18819 18838 1119 546944 n/a n/aGCCACTAACCCACCTCTTAA 5-10-5 42 19140 19159 1120 546946 n/a n/aACTCCCATCTACTCCCCCAT 5-10-5 41 19291 19310 1121 546954 n/a n/aCTGCTGATTGTGTCTGGCTC 5-10-5 71 20235 20254 1122 546955 n/a n/aACAAGGCTTCGAGGACAGCC 5-10-5 49 20339 20358 1123 546964 n/a n/aGCGATTCCTTGCCTCTGCTG 5-10-5 53 21550 21569 1124 546967 n/a n/aCACCGCGCGAATGCCTGCCT 5-10-5 93 22657 22676 1125 546969 n/a n/aATCCAACCTCTCTCCCTATC 5-10-5 53 22901 22920 1126 546970 n/a n/aGCCCAAGCCTACATGCATAC 5-10-5 61 23426 23445 1127 546975 n/a n/aGGCCTGGATACAGCCTTTCT 5-10-5 70 23825 23844 1128 546977 n/a n/aGTCCCGAAGAGTCAAGTCCA 5-10-5 76 24253 24272 1129 546979 n/a n/aACTGTTGTCCATAGCAGCAT 5-10-5 71 24504 24523 1130 546980 n/a n/aAGCCCTCAATTGTTGCTGGT 5-10-5 79 24664 24683 1131 546983 n/a n/aGATGACCTGCAGATGCACAG 5-10-5 74 24978 24997 1132 546986 n/a n/aCAGGATAGAACTGATGGTCC 5-10-5 91 25318 25337 1133 546990 n/a n/aAGAACAGGAGACAATCCACT 5-10-5 49 25680 25699 1134 546994 n/a n/aGTTCATGTGGCAACCTGTGA 5-10-5 58 26112 26131 1135 547677 n/a n/aCATCACTGTCCTATAACTAT 5-10-5 62 16105 16124 1136 547678 n/a n/aTACCTATATCACTGTAACTA 5-10-5 21 16125 16144 1137 547679 n/a n/aTGTACCTATATCACTGTAAC 5-10-5 28 16127 16146 1138 547680 n/a n/aTATCACTGTACCTATATCAC 5-10-5 41 16133 16152 1139 547681 n/a n/aAATATCACTGTACCTATATC 5-10-5 6 16135 16154 1140 547682 n/a n/aCAATATCACTGTACCTATAT 5-10-5 20 16136 16155 1141 547683 n/a n/aACTATATCACTGTCCTATAA 5-10-5 33 16162 16181 1142 547684 n/a n/aTAACTATATCACTGTCCTAT 5-10-5 43 16164 16183 1143 547685 n/a n/aATAACTATATCACTGTCCTA 5-10-5 35 16165 16184 1144 547686 n/a n/aCTGTCCTATAACTATATCAC 5-10-5 36 16172 16191 1145 547687 n/a n/aACTGTCCTATAACTATATCA 5-10-5 41 16173 16192 1146 547688 n/a n/aCACTGTCCTATAACTATATC 5-10-5 47 16174 16193 1147 547689 n/a n/aGTAACAATATCACTGTCCTA 5-10-5 73 16184 16203 1148 547690 n/a n/aCTGTAACAATATCACTGTCC 5-10-5 76 16186 16205 1149 547691 n/a n/aACTGTAACAATATCACTGTC 5-10-5 36 16187 16206 1150 547692 n/a n/aCACTGTACCTATAACTGTAA 5-10-5 47 16200 16219 1151 547693 n/a n/aTCACTGTACCTATAACTGTA 5-10-5 61 16201 16220 1152 547694 n/a n/aGTCACTGTACCTATAACTGT 5-10-5 92 16202 16221 1153 547695 n/a n/aACTGTCCTATAACTATTACA 5-10-5 31 16224 16243 1154 547696 n/a n/aCACTGTCCTATAACTATTAC 5-10-5 26 16225 16244 1155 547697 n/a n/aTCACTGTCCTATAACTATTA 5-10-5 63 16226 16245 1156 547698 n/a n/aACCTATAACTATAACAATAT 5-10-5 0 16245 16264 1157 547699 n/a n/aTACCTATAACTATAACAATA 5-10-5 10 16246 16265 1158 547700 n/a n/aGTACCTATAACTATAACAAT 5-10-5 0 16247 16266 1159 547701 n/a n/aCATCACTGTACCTATAACTA 5-10-5 49 16254 16273 1160 547702 n/a n/aACATCACTGTACCTATAACT 5-10-5 44 16255 16274 1161 547703 n/a n/aCAACATCACTGTACCTATAA 5-10-5 25 16257 16276 1162 547704 n/a n/aACATCTTGTCATTAACATCC 5-10-5 61 16435 16454 1163 547705 n/a n/aGCACCCAATACAGGGCCAGG 5-10-5 69 16512 16531 1164 547706 n/a n/aTGCCTCCTGGCAGCCTTCAA 5-10-5 73 16694 16713 1165 547707 n/a n/aTGAAAAGCCACGCCCTTAGC 5-10-5 32 16975 16994 1166 547708 n/a n/aGCCAGGAGACAGCCCTACTC 5-10-5 67 17055 17074 1167 547709 n/a n/aAGCCCAATGTCCTAACCTGT 5-10-5 76 17791 17810 1168 547710 n/a n/aTGCGGTTATATGGGCTGAAG 5-10-5 85 19540 19559 1169 547711 n/a n/aCCTTTAGCCACTCCTCTTGC 5-10-5 45 20061 20080 1170 547712 n/a n/aCCCCATGGTACCAAAGCCAT 5-10-5 79 20528 20547 1171 547713 n/a n/aCTCAATGCCACCCTTTCCCC 5-10-5 37 20880 20899 1172 547714 n/a n/aCTGTCTAACTGGCCTGGCTG 5-10-5 19 21326 21345 1173 547715 n/a n/aGGTCAGAAGGCCTCTTATTC 5-10-5 21 21750 21769 1174 547716 n/a n/aCCATCTGTCCCCTCAATCCC 5-10-5 9 22197 22216 1175 547717 n/a n/aACTCTGGCACTGGTCATGGA 5-10-5 54 22761 22780 1176 547718 n/a n/aATAAAGTGCGATTAAGCCCC 5-10-5 86 23515 23534 1177 547719 n/a n/aTACCAAGCTTGTAGAAGGGA 5-10-5 69 23633 23652 1178 547720 n/a n/aGAAAGACGGCCAATGGGAAA 5-10-5 8 24177 24196 1179 547721 n/a n/aCTCTATCAAAATCCTGCTGC 5-10-5 68 25527 25546 1180 547722 n/a n/aCTCCAGTCACCACCATTGCC 5-10-5 80 25860 25879 1181

TABLE 17 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 SEQ ISISStart Stop % Start Stop ID NO Site Site Sequence Motif inhibition SiteSite NO 531231 n/a n/a TATCACTGTACTAGTTTCCT 5-10-5 91 14744 14763 33414815 14834 14886 14905 14945 14964 15005 15024 15077 15096 15220 1523915292 15311 15351 15370 15411 15430 15483 15502 15555 15574 15613 1563215685 15704 15815 15834 15887 15906 15945 15964 546599 n/a n/aAAGAGTAAGCCTTCACAGGG 5-10-5 82 27583 27602 1182 546606 n/a n/aCTCACCAGAGTTGTCCCCAG 5-10-5 0 27722 27741 1183 546999 n/a n/aGCAGCTCACACCCAAAAAGC 5-10-5 29 27004 27023 1184 547000 n/a n/aTCTGTTACCTTGAGGATTGT 5-10-5 63 27276 27295 1185 547006 n/a n/aCGCCATCTGCCCTGTACAGA 5-10-5 39 28248 28267 1186 547008 n/a n/aTTGGTGGTGGGATTGGTGGT 5-10-5 81 28333 28352 1187 28388 28407 28443 2846228608 28627 28620 28639 547009 n/a n/a AATTGGTGGTGGGATTGGTG 5-10-5 7328335 28354 1188 547010 n/a n/a GAATTGGTGGTGGGATTGGT 5-10-5 39 2833628355 1189 547011 n/a n/a GGCAGGATTGGTGGTGGAAT 5-10-5 22 28352 283711190 547013 n/a n/a TGAGATTGGTGGTGGGTGGC 5-10-5 0 28369 28388 1191547015 n/a n/a GGTGGTGGGATTGGTGCTGA 5-10-5 55 28429 28448 1192 547016n/a n/a GTAGGTGGTGGGATTGGTGG 5-10-5 62 28456 28475 1193 28535 28554547017 n/a n/a GGTAGGTGGTGGGATTGGTG 5-10-5 61 28457 28476 1194 2853628555 547018 n/a n/a GGTGGCGGGATTGGTGGTGG 5-10-5 58 28477 28496 119528556 28575 547019 n/a n/a GATCGGTGGTGGGATTGGTC 5-10-5 83 28500 285191196 28579 28598 547020 n/a n/a GGATCGGTGGTGGGATTGGT 5-10-5 47 2850128520 1197 28580 28599 547021 n/a n/a TTGGTGGCGGGATCGGTGGT 5-10-5 5728510 28529 1198 28589 28608 547022 n/a n/a ATTGGTGGCGGGATCGGTGG 5-10-569 28511 28530 1199 547023 n/a n/a GATTGGTGGCGGGATCGGTG 5-10-5 91 2851228531 1200 547024 n/a n/a GGATTGGTGGCGGGATCGGT 5-10-5 56 28513 285321201 547025 n/a n/a TGGTGGTGGGATTGGTGGTT 5-10-5 72 28607 28626 1202547029 n/a n/a TCTTCTAGGGCCACACCTCT 5-10-5 50 28891 28910 1203 547035n/a n/a TGGTCCCAAATTGGAGTGCA 5-10-5 40 29383 29402 1204 547039 n/a n/aTCTCTATACAGCTGGGCACA 5-10-5 0 29997 30016 1205 547049 n/a n/aCACTTCCCAGCAACCCTCAC 5-10-5 20 30765 30784 1206 547055 n/a n/aGCTCCTGGCAGCAATGACCC 5-10-5 70 31104 31123 1207 547059 n/a n/aGGGTATCTTCACTGTTCCAG 5-10-5 12 31540 31559 1208 547063 n/a n/aCGTCATGCTTACCTTTCTCC 5-10-5 23 31955 31974 1209 547069 n/a n/aGCCCTCCGAGCTTTGGCAAC 5-10-5 35 32581 32600 1210 547071 n/a n/aGCAGCCCCCCAGAAATCCCA 5-10-5 27 32708 32727 1211 547076 n/a n/aTCTCAAGCAGCCTATTGTGT 5-10-5 14 33263 33282 1212 547080 n/a n/aGTGCAAGACCTTGCTTGCCA 5-10-5 54 33657 33676 1213 547081 n/a n/aCTGTAGTCCACTACACAGCA 5-10-5 83 33801 33820 1214 547082 n/a n/aTCTCCCTGAGTCACAGTGGA 5-10-5 64 33881 33900 1215 547085 n/a n/aCCAGGTGCAGCACGGAGAGG 5-10-5 44 34479 34498 1216 547723 n/a n/aTAGAATGGCAGGGTTCTGTG 5-10-5 53 27357 27376 1217 547724 n/a n/aGATGCATCCAACACTTACCC 5-10-5 16 28059 28078 1218 547725 n/a n/aATTGGTGGTGGGATTGGTGG 5-10-5 26 28334 28353 1219 28389 28408 28444 2846328523 28542 28609 28628 28621 28640 547726 n/a n/a GCAGGATTGGTGGTGGAATT5-10-5 0 28351 28370 1220 547727 n/a n/a TGGCAGGATTGGTGGTGGAA 5-10-5 028353 28372 1221 547728 n/a n/a GAGATTGGTGGTGGGTGGCA 5-10-5 88 2836828387 1222 547729 n/a n/a GTGAGATTGGTGGTGGGTGG 5-10-5 45 28370 283891223 547730 n/a n/a GATTGGTGGTGGGATTGGTG 5-10-5 60 28390 28409 122428433 28452 28445 28464 28524 28543 28610 28629 28622 28641 547731 n/an/a GGATTGGTGGTGGGATTGGT 5-10-5 49 28391 28410 1225 28434 28453 2844628465 28525 28544 28611 28630 28623 28642 547732 n/a n/aAGGATTGGTGGTGGGATTGG 5-10-5 0 28392 28411 1226 547733 n/a n/aTAGGATTGGTGGTGGGATTG 5-10-5 0 28393 28412 1227 547734 n/a n/aGTAGGATTGGTGGTGGGATT 5-10-5 14 28394 28413 1228 547735 n/a n/aGGTAGGATTGGTGGTGGGAT 5-10-5 39 28395 28414 1229 547736 n/a n/aTGGTAGGATTGGTGGTGGGA 5-10-5 54 28396 28415 1230 547737 n/a n/aTGGTGGTGGGATTGGTGCTG 5-10-5 59 28430 28449 1231 547738 n/a n/aTTGGTGGTGGGATTGGTGCT 5-10-5 41 28431 28450 1232 547739 n/a n/aATTGGTGGTGGGATTGGTGC 5-10-5 12 28432 28451 1233 547740 n/a n/aAGGTGGTGGGATTGGTGGTG 5-10-5 30 28454 28473 1234 28533 28552 547741 n/an/a TAGGTGGTGGGATTGGTGGT 5-10-5 47 28455 28474 1235 28534 28553 547742n/a n/a ATCGGTGGTGGGATTGGTCG 5-10-5 57 28499 28518 1236 28578 28597547743 n/a n/a GGTGGTGGGATTGGTGGCGG 5-10-5 61 28520 28539 1237 547744n/a n/a TGGTGGTGGGATTGGTGGCG 5-10-5 65 28521 28540 1238 547745 n/a n/aTTGGTGGTGGGATTGGTGGC 5-10-5 55 28522 28541 1239 547746 n/a n/aGTTGGTGGCGGGATCGGTGG 5-10-5 0 28590 28609 1240 547748 n/a n/aGGTTGGTGGCGGGATCGGTG 5-10-5 78 28591 28610 1241 547750 n/a n/aTGGTTGGTGGCGGGATCGGT 5-10-5 41 28592 28611 1242 547752 n/a n/aGTGGTTGGTGGCGGGATCGG 5-10-5 41 28593 28612 1243 547754 n/a n/aGGGATTGGTGGTTGGTGGCG 5-10-5 47 28600 28619 1244 547756 n/a n/aGGGTCTTGCTCCACCCACAT 5-10-5 49 29244 29263 1245 547758 n/a n/aCCAAGTAGTGCAAGGCATGT 5-10-5 24 29540 29559 1246 547760 n/a n/aATCATGCTTACTGCAAGTGA 5-10-5 19 30219 30238 1247 547762 n/a n/aTGAAACTGGGCAGTCCTTCC 5-10-5 0 30417 30436 1248 547764 n/a n/aCCACCTTCTTACATATGCTA 5-10-5 24 30644 30663 1249 547766 n/a n/aGCCTCTCAGACGGCACAGAC 5-10-5 0 30902 30921 1250 547768 n/a n/aTTGCCCTCACACATTCGAAT 5-10-5 0 30977 30996 1251 547770 n/a n/aTGCTTTCTGCCCAACCTCTA 5-10-5 48 31727 31746 1252 547772 n/a n/aCTGTGCTCCCGGCCATTAGC 5-10-5 0 32312 32331 1253 547774 n/a n/aGAGACAGTTTGGCAAGCTAC 5-10-5 46 32389 32408 1254 547776 n/a n/aGGAGAGAGACGGCACCCTGT 5-10-5 48 32828 32847 1255 547778 n/a n/aTCACCTGTGAGTAACCAATA 5-10-5 53 33085 33104 1256 547780 n/a n/aCCCCTCTTAAATAGCACATG 5-10-5 67 33441 33460 1257 547782 n/a n/aCCAAGTATCTCATGTGCCTG 5-10-5 67 33580 33599 1258

TABLE 18 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 ISISStart Stop % Start Stop SEQ NO Site Site Sequence Motif inhibition SiteSite ID NO 531231 n/a n/a TATCACTGTACTAGTTTCCT 5-10-5 90 14744 14763 33414815 14834 14886 14905 14945 14964 15005 15024 15077 15096 15220 1523915292 15311 15351 15370 15411 15430 15483 15502 15555 15574 15613 1563215685 15704 15815 15834 15887 15906 15945 15964 548706 n/a n/aCTAGTTTCCTATAACT 3-10-3 0 14738 14753 1259 14809 14824 14880 14895 1493914954 15071 15086 15214 15229 15286 15301 15345 15360 15477 15492 1554915564 15607 15622 15679 15694 15750 15765 15809 15824 15881 15896 1593915954 548707 n/a n/a ACTAGTTTCCTATAAC 3-10-3 10 14739 14754 1260 1481014825 14881 14896 14940 14955 15000 15015 15072 15087 15215 15230 1528715302 15346 15361 15406 15421 15478 15493 15550 15565 15608 15623 1568015695 15751 15766 15810 15825 15882 15897 15940 15955 548708 n/a n/aTACTAGTTTCCTATAA 3-10-3 0 14740 14755 1261 14811 14826 14882 14897 1494114956 15001 15016 15073 15088 15216 15231 15288 15303 15347 15362 1540715422 15479 15494 15551 15566 15609 15624 15681 15696 15752 15767 1581115826 15883 15898 15941 15956 548709 n/a n/a GTACTAGTTTCCTATA 3-10-3 014741 14756 1262 14812 14827 14883 14898 14942 14957 15002 15017 1507415089 15217 15232 15289 15304 15348 15363 15408 15423 15480 15495 1555215567 15610 15625 15682 15697 15753 15768 15812 15827 15884 15899 1594215957 548710 n/a n/a TGTACTAGTTTCCTAT 3-10-3 0 14742 14757 1263 1481314828 14884 14899 14943 14958 15003 15018 15075 15090 15218 15233 1529015305 15349 15364 15409 15424 15481 15496 15553 15568 15611 15626 1568315698 15813 15828 15885 15900 15943 15958 548711 n/a n/aCTGTACTAGTTTCCTA 3-10-3 21 14743 14758 1264 14814 14829 14885 1490014944 14959 15004 15019 15076 15091 15219 15234 15291 15306 15350 1536515410 15425 15482 15497 15554 15569 15612 15627 15684 15699 15814 1582915886 15901 15944 15959 548712 n/a n/a ACTGTACTAGTTTCCT 3-10-3 9 1474414759 1265 14815 14830 14886 14901 14945 14960 15005 15020 15077 1509215220 15235 15292 15307 15351 15366 15411 15426 15483 15498 15555 1557015613 15628 15685 15700 15815 15830 15887 15902 15945 15960 548713 n/an/a CACTGTACTAGTTTCC 3-10-3 33 14745 14760 1266 14816 14831 14887 1490214946 14961 15006 15021 15078 15093 15221 15236 15293 15308 15352 1536715412 15427 15484 15499 15556 15571 15614 15629 15686 15701 15816 1583115888 15903 15946 15961 548714 n/a n/a TCACTGTACTAGTTTC 3-10-3 15 1474614761 1267 14817 14832 14888 14903 14947 14962 15007 15022 15079 1509415222 15237 15294 15309 15353 15368 15413 15428 15485 15500 15557 1557215615 15630 15687 15702 15817 15832 15889 15904 15947 15962 548715 n/an/a ATCACTGTACTAGTTT 3-10-3 0 14747 14762 1268 14818 14833 14889 1490414948 14963 15008 15023 15080 15095 15152 15167 15223 15238 15295 1531015354 15369 15414 15429 15486 15501 15558 15573 15616 15631 15688 1570315818 15833 15890 15905 15948 15963 548716 n/a n/a TATCACTGTACTAGTT3-10-3 10 14748 14763 1269 14819 14834 14890 14905 14949 14964 1500915024 15081 15096 15153 15168 15224 15239 15296 15311 15355 15370 1541515430 15487 15502 15559 15574 15617 15632 15689 15704 15819 15834 1589115906 15949 15964 548717 n/a n/a ACTAGTTTCCTATAACT 3-10-4 0 14738 147541270 14809 14825 14880 14896 14939 14955 15071 15087 15214 15230 1528615302 15345 15361 15477 15493 15549 15565 15607 15623 15679 15695 1575015766 15809 15825 15881 15897 15939 15955 548718 n/a n/aTACTAGTTTCCTATAAC 3-10-4 0 14739 14755 1271 14810 14826 14881 1489714940 14956 15000 15016 15072 15088 15215 15231 15287 15303 15346 1536215406 15422 15478 15494 15550 15566 15608 15624 15680 15696 15751 1576715810 15826 15882 15898 15940 15956 548719 n/a n/a GTACTAGTTTCCTATAA3-10-4 0 14740 14756 1272 14811 14827 14882 14898 14941 14957 1500115017 15073 15089 15216 15232 15288 15304 15347 15363 15407 15423 1547915495 15551 15567 15609 15625 15681 15697 15752 15768 15811 15827 1588315899 15941 15957 548720 n/a n/a TGTACTAGTTTCCTATA 3-10-4 0 14741 147571273 14812 14828 14883 14899 14942 14958 15002 15018 15074 15090 1521715233 15289 15305 15348 15364 15408 15424 15480 15496 15552 15568 1561015626 15682 15698 15812 15828 15884 15900 15942 15958 548721 n/a n/aCTGTACTAGTTTCCTAT 3-10-4 27 14742 14758 1274 14813 14829 14884 1490014943 14959 15003 15019 15075 15091 15218 15234 15290 15306 15349 1536515409 15425 15481 15497 15553 15569 15611 15627 15683 15699 15813 1582915885 15901 15943 15959 548722 n/a n/a ACTGTACTAGTTTCCTA 3-10-4 26 1474314759 1275 14814 14830 14885 14901 14944 14960 15004 15020 15076 1509215219 15235 15291 15307 15350 15366 15410 15426 15482 15498 15554 1557015612 15628 15684 15700 15814 15830 15886 15902 15944 15960 548723 n/an/a CACTGTACTAGTTTCCT 3-10-4 62 14744 14760 1276 14815 14831 14886 1490214945 14961 15005 15021 15077 15093 15220 15236 15292 15308 15351 1536715411 15427 15483 15499 15555 15571 15613 15629 15685 15701 15815 1583115887 15903 15945 15961 548724 n/a n/a TCACTGTACTAGTTTCC 3-10-4 61 1474514761 1277 14816 14832 14887 14903 14946 14962 15006 15022 15078 1509415221 15237 15293 15309 15352 15368 15412 15428 15484 15500 15556 1557215614 15630 15686 15702 15816 15832 15888 15904 15946 15962 548725 n/an/a ATCACTGTACTAGTTTC 3-10-4 32 14746 14762 1278 14817 14833 14888 1490414947 14963 15007 15023 15079 15095 15222 15238 15294 15310 15353 1536915413 15429 15485 15501 15557 15573 15615 15631 15687 15703 15817 1583315889 15905 15947 15963 548726 n/a n/a TATCACTGTACTAGTTT 3-10-4 21 1474714763 1279 14818 14834 14889 14905 14948 14964 15008 15024 15080 1509615152 15168 15223 15239 15295 15311 15354 15370 15414 15430 15486 1550215558 15574 15616 15632 15688 15704 15818 15834 15890 15906 15948 15964548727 n/a n/a ACTAGTTTCCTATAACT 4-10-3 0 14738 14754 1270 14809 1482514880 14896 14939 14955 15071 15087 15214 15230 15286 15302 15345 1536115477 15493 15549 15565 15607 15623 15679 15695 15750 15766 15809 1582515881 15897 15939 15955 548728 n/a n/a TACTAGTTTCCTATAAC 4-10-3 0 1473914755 1271 14810 14826 14881 14897 14940 14956 15000 15016 15072 1508815215 15231 15287 15303 15346 15362 15406 15422 15478 15494 15550 1556615608 15624 15680 15696 15751 15767 15810 15826 15882 15898 15940 15956548729 n/a n/a GTACTAGTTTCCTATAA 4-10-3 13 14740 14756 1272 14811 1482714882 14898 14941 14957 15001 15017 15073 15089 15216 15232 15288 1530415347 15363 15407 15423 15479 15495 15551 15567 15609 15625 15681 1569715752 15768 15811 15827 15883 15899 15941 15957 548730 n/a n/aTGTACTAGTTTCCTATA 4-10-3 0 14741 14757 1273 14812 14828 14883 1489914942 14958 15002 15018 15074 15090 15217 15233 15289 15305 15348 1536415408 15424 15480 15496 15552 15568 15610 15626 15682 15698 15812 1582815884 15900 15942 15958 548731 n/a n/a CTGTACTAGTTTCCTAT 4-10-3 49 1474214758 1274 14813 14829 14884 14900 14943 14959 15003 15019 15075 1509115218 15234 15290 15306 15349 15365 15409 15425 15481 15497 15553 1556915611 15627 15683 15699 15813 15829 15885 15901 15943 15959 548732 n/an/a ACTGTACTAGTTTCCTA 4-10-3 36 14743 14759 1275 14814 14830 14885 1490114944 14960 15004 15020 15076 15092 15219 15235 15291 15307 15350 1536615410 15426 15482 15498 15554 15570 15612 15628 15684 15700 15814 1583015886 15902 15944 15960 548733 n/a n/a CACTGTACTAGTTTCCT 4-10-3 84 1474414760 1276 14815 14831 14886 14902 14945 14961 15005 15021 15077 1509315220 15236 15292 15308 15351 15367 15411 15427 15483 15499 15555 1557115613 15629 15685 15701 15815 15831 15887 15903 15945 15961 548734 n/an/a TCACTGTACTAGTTTCC 4-10-3 51 14745 14761 1277 14816 14832 14887 1490314946 14962 15006 15022 15078 15094 15221 15237 15293 15309 15352 1536815412 15428 15484 15500 15556 15572 15614 15630 15686 15702 15816 1583215888 15904 15946 15962 548735 n/a n/a ATCACTGTACTAGTTTC 4-10-3 48 1474614762 1278 14817 14833 14888 14904 14947 14963 15007 15023 15079 1509515222 15238 15294 15310 15353 15369 15413 15429 15485 15501 15557 1557315615 15631 15687 15703 15817 15833 15889 15905 15947 15963 548736 n/an/a TATCACTGTACTAGTTT 4-10-3 21 14747 14763 1279 14818 14834 14889 1490514948 14964 15008 15024 15080 15096 15152 15168 15223 15239 15295 1531115354 15370 15414 15430 15486 15502 15558 15574 15616 15632 15688 1570415818 15834 15890 15906 15948 15964 548737 n/a n/a ACTAGTTTCCTATAACT4-9-4 11 14738 14754 1270 14809 14825 14880 14896 14939 14955 1507115087 15214 15230 15286 15302 15345 15361 15477 15493 15549 15565 1560715623 15679 15695 15750 15766 15809 15825 15881 15897 15939 15955 548738n/a n/a TACTAGTTTCCTATAAC 4-9-4 0 14739 14755 1271 14810 14826 1488114897 14940 14956 15000 15016 15072 15088 15215 15231 15287 15303 1534615362 15406 15422 15478 15494 15550 15566 15608 15624 15680 15696 1575115767 15810 15826 15882 15898 15940 15956 548739 n/a n/aGTACTAGTTTCCTATAA 4-9-4 0 14740 14756 1272 14811 14827 14882 14898 1494114957 15001 15017 15073 15089 15216 15232 15288 15304 15347 15363 1540715423 15479 15495 15551 15567 15609 15625 15681 15697 15752 15768 1581115827 15883 15899 15941 15957 548740 n/a n/a TGTACTAGTTTCCTATA 4-9-4 014741 14757 1273 14812 14828 14883 14899 14942 14958 15002 15018 1507415090 15217 15233 15289 15305 15348 15364 15408 15424 15480 15496 1555215568 15610 15626 15682 15698 15812 15828 15884 15900 15942 15958 548741n/a n/a CTGTACTAGTTTCCTAT 4-9-4 69 14742 14758 1274 14813 14829 1488414900 14943 14959 15003 15019 15075 15091 15218 15234 15290 15306 1534915365 15409 15425 15481 15497 15553 15569 15611 15627 15683 15699 1581315829 15885 15901 15943 15959 548742 n/a n/a ACTGTACTAGTTTCCTA 4-9-4 5014743 14759 1275 14814 14830 14885 14901 14944 14960 15004 15020 1507615092 15219 15235 15291 15307 15350 15366 15410 15426 15482 15498 1555415570 15612 15628 15684 15700 15814 15830 15886 15902 15944 15960 548743n/a n/a CACTGTACTAGTTTCCT 4-9-4 80 14744 14760 1276 14815 14831 1488614902 14945 14961 15005 15021 15077 15093 15220 15236 15292 15308 1535115367 15411 15427 15483 15499 15555 15571 15613 15629 15685 15701 1581515831 15887 15903 15945 15961 548744 n/a n/a TCACTGTACTAGTTTCC 4-9-4 8314745 14761 1277 14816 14832 14887 14903 14946 14962 15006 15022 1507815094 15221 15237 15293 15309 15352 15368 15412 15428 15484 15500 1555615572 15614 15630 15686 15702 15816 15832 15888 15904 15946 15962 548745n/a n/a ATCACTGTACTAGTTTC 4-9-4 71 14746 14762 1278 14817 14833 1488814904 14947 14963 15007 15023 15079 15095 15222 15238 15294 15310 1535315369 15413 15429 15485 15501 15557 15573 15615 15631 15687 15703 1581715833 15889 15905 15947 15963 548746 n/a n/a TATCACTGTACTAGTTT 4-9-4 4014747 14763 1279 14818 14834 14889 14905 14948 14964 15008 15024 1508015096 15152 15168 15223 15239 15295 15311 15354 15370 15414 15430 1548615502 15558 15574 15616 15632 15688 15704 15818 15834 15890 15906 1594815964 548747 n/a n/a TACTAGTTTCCTATAACT 4-10-4 2 14738 14755 1280 1480914826 14880 14897 14939 14956 15071 15088 15214 15231 15286 15303 1534515362 15477 15494 15549 15566 15607 15624 15679 15696 15750 15767 1580915826 15881 15898 15939 15956 548748 n/a n/a GTACTAGTTTCCTATAAC 4-10-4 014739 14756 1281 14810 14827 14881 14898 14940 14957 15000 15017 1507215089 15215 15232 15287 15304 15346 15363 15406 15423 15478 15495 1555015567 15608 15625 15680 15697 15751 15768 15810 15827 15882 15899 1594015957 548749 n/a n/a TGTACTAGTTTCCTATAA 4-10-4 0 14740 14757 1282 1481114828 14882 14899 14941 14958 15001 15018 15073 15090 15216 15233 1528815305 15347 15364 15407 15424 15479 15496 15551 15568 15609 15626 1568115698 15811 15828 15883 15900 15941 15958 548750 n/a n/aCTGTACTAGTTTCCTATA 4-10-4 62 14741 14758 1283 14812 14829 14883 1490014942 14959 15002 15019 15074 15091 15217 15234 15289 15306 15348 1536515408 15425 15480 15497 15552 15569 15610 15627 15682 15699 15812 1582915884 15901 15942 15959 548751 n/a n/a ACTGTACTAGTTTCCTAT 4-10-4 5314742 14759 1284 14813 14830 14884 14901 14943 14960 15003 15020 1507515092 15218 15235 15290 15307 15349 15366 15409 15426 15481 15498 1555315570 15611 15628 15683 15700 15813 15830 15885 15902 15943 15960 548752n/a n/a CACTGTACTAGTTTCCTA 4-10-4 89 14743 14760 1285 14814 14831 1488514902 14944 14961 15004 15021 15076 15093 15219 15236 15291 15308 1535015367 15410 15427 15482 15499 15554 15571 15612 15629 15684 15701 1581415831 15886 15903 15944 15961 548753 n/a n/a TCACTGTACTAGTTTCCT 4-10-482 14744 14761 1286 14815 14832 14886 14903 14945 14962 15005 1502215077 15094 15220 15237 15292 15309 15351 15368 15411 15428 15483 1550015555 15572 15613 15630 15685 15702 15815 15832 15887 15904 15945 15962548754 n/a n/a ATCACTGTACTAGTTTCC 4-10-4 77 14745 14762 1287 14816 1483314887 14904 14946 14963 15006 15023 15078 15095 15221 15238 15293 1531015352 15369 15412 15429 15484 15501 15556 15573 15614 15631 15686 1570315816 15833 15888 15905 15946 15963 548755 n/a n/a TATCACTGTACTAGTTTC4-10-4 20 14746 14763 1288 14817 14834 14888 14905 14947 14964 1500715024 15079 15096 15222 15239 15294 15311 15353 15370 15413 15430 1548515502 15557 15574 15615 15632 15687 15704 15817 15834 15889 15906 1594715964 548756 n/a n/a GTATCACTGTACTAGTT 4-9-4 81 14748 14764 1289 1481914835 14890 14906 14949 14965 15009 15025 15081 15097 15153 15169 1522415240 15296 15312 15355 15371 15415 15431 15487 15503 15559 15575 1561715633 15689 15705 15819 15835 15891 15907 15949 15965 548757 n/a n/aAGTATCACTGTACTAGT 4-9-4 87 14749 14765 1290 14820 14836 14891 1490714950 14966 15010 15026 15082 15098 15154 15170 15225 15241 15297 1531315356 15372 15416 15432 15488 15504 15560 15576 15618 15634 15690 1570615820 15836 15892 15908 15950 15966 548758 n/a n/a CAGTATCACTGTACTAG4-9-4 97 14750 14766 1291 14821 14837 14892 14908 14951 14967 1501115027 15083 15099 15155 15171 15226 15242 15298 15314 15357 15373 1541715433 15489 15505 15561 15577 15619 15635 15691 15707 15821 15837 1589315909 15951 15967 548759 n/a n/a AACAGTATCACTGTACT 4-9-4 68 14752 147681292 14823 14839 14894 14910 14953 14969 15013 15029 15085 15101 1515715173 15228 15244 15300 15316 15359 15375 15419 15435 15491 15507 1556315579 15621 15637 15693 15709 15823 15839 15895 15911 15953 15969 548760n/a n/a TAACAGTATCACTGTAC 4-9-4 53 14753 14769 1293 14824 14840 1489514911 14954 14970 15014 15030 15086 15102 15158 15174 15229 15245 1530115317 15360 15376 15420 15436 15492 15508 15564 15580 15622 15638 1569415710 15824 15840 15896 15912 15954 15970 548761 n/a n/aCTAACAGTATCACTGTA 4-9-4 49 14754 14770 1294 14825 14841 14896 1491215015 15031 15087 15103 15230 15246 15302 15318 15421 15437 15493 1550915623 15639 15825 15841 15955 15971 548762 n/a n/a TCTAACAGTATCACTGT4-9-4 16 14755 14771 1295 14826 14842 14897 14913 15016 15032 1508815104 15231 15247 15303 15319 15422 15438 15494 15510 15624 15640 1582615842 15956 15972 548763 n/a n/a CTCTAACAGTATCACTG 4-9-4 44 14756 147721296 14827 14843 14898 14914 15017 15033 15089 15105 15232 15248 1530415320 15423 15439 15495 15511 15625 15641 15827 15843 15957 15973 548764n/a n/a TATCACTGTCCTATAAC 4-9-4 31 14772 14788 1297 14843 14859 1517715193 15583 15599 15713 15729 16012 16028 16083 16099 16161 16177 1618016196 16231 16247 548765 n/a n/a ATATCACTGTCCTATAA 4-9-4 0 14773 147891298 14844 14860 15178 15194 15584 15600 15714 15730 16013 16029 1608416100 16162 16178 16181 16197 16232 16248 548766 n/a n/aTATATCACTGTCCTATA 4-9-4 36 14774 14790 1299 14845 14861 15179 1519515715 15731 16163 16179 548767 n/a n/a TATCACTGTCCTATATC 4-9-4 59 1478514801 1300 14856 14872 14981 14997 15119 15135 15190 15206 15262 1527815387 15403 15525 15541 15655 15671 15726 15742 15857 15873 15987 16003548768 n/a n/a GTATCACTGTCCTATAT 4-9-4 56 14786 14802 1301 14982 1499815120 15136 15388 15404 15526 15542 15988 16004 548769 n/a n/aAGTATCACTGTCCTATA 4-9-4 64 14787 14803 1302 14983 14999 15121 1513715389 15405 15527 15543 15989 16005 548770 n/a n/a TAACAGTATCACTGTCC4-9-4 92 14791 14807 1303 14987 15003 15053 15069 15125 15141 1539315409 15459 15475 15531 15547 15993 16009 548771 n/a n/aATAACAGTATCACTGTC 4-9-4 62 14792 14808 1304 14988 15004 15054 1507015126 15142 15394 15410 15460 15476 15532 15548 15994 16010 548772 n/an/a TATAACAGTATCACTGT 4-9-4 0 14793 14809 1305 14989 15005 15055 1507115127 15143 15160 15176 15362 15378 15395 15411 15461 15477 15533 1554915566 15582 15696 15712 15898 15914 15995 16011 548773 n/a n/aCTATAACAGTATCACTG 4-9-4 0 14794 14810 1306 14990 15006 15056 15072 1512815144 15161 15177 15363 15379 15396 15412 15462 15478 15534 15550 1556715583 15697 15713 15899 15915 15996 16012 548774 n/a n/aCCTATAACTATAACAGT 4-9-4 0 14801 14817 1307 15063 15079 15168 15184 1546915485 15541 15557 15574 15590 15704 15720 15775 15791 16003 16019 548775n/a n/a TCCTATAACTATAACAG 4-9-4 0 14802 14818 1308 15064 15080 1516915185 15470 15486 15542 15558 15575 15591 15705 15721 16004 16020 548776n/a n/a CCTATAACTATAACAAT 4-9-4 0 14872 14888 1309 14931 14947 1520615222 15278 15294 15337 15353 15599 15615 15671 15687 15742 15758 1580115817 15873 15889 15931 15947 16074 16090 16099 16115 16152 16168 1624716263 548777 n/a n/a GTAACAGTATCACTGTA 4-9-4 41 14955 14971 1310 548778n/a n/a ATAACAGTATCACTGTA 4-9-4 20 15159 15175 1311 15361 15377 1556515581 15695 15711 15897 15913 548779 n/a n/a GTCCTATAACTATAACA 4-9-4 015170 15186 1312 15576 15592 15706 15722 16005 16021 16076 16092 1610116117 16154 16170 548780 n/a n/a TGTCCTATAACTATAAC 4-9-4 22 15171 151871313 15577 15593 15707 15723 16006 16022 16077 16093 16102 16118 1615516171 548781 n/a n/a ACCTATAACTATAACAG 4-9-4 0 15776 15792 1314 548782n/a n/a TACCTATAACTATAACA 4-9-4 0 15777 15793 1315 16249 16265 548783n/a n/a ACCTATAACTATAACAA 4-9-4 0 16248 16264 1316

Example 3: Antisense Inhibition of Human PKK in HepaRG™ Cells byAntisense Oligonucleotides with MOE, Deoxy and cEt Sugar Modifications

Additional antisense oligonucleotides were designed targeting a PKKnucleic acid and were tested for their effects on PKK mRNA in vitro.

The chimeric antisense oligonucleotides in the tables below weredesigned as deoxy, MOE and cEt gapmers. The gapmers are 16 nucleosidesin length wherein the nucleoside have either a MOE sugar modification, acEt sugar modification, or a deoxy modification. The ‘Chemistry’ columndescribes the sugar modifications of each oligonucleotide. ‘k’ indicatesan cEt sugar modification; the number indicates the number ofdeoxynucleosides; otherwise, ‘d’ indicates a deoxynucleoside; and ‘e’indicates a 2′-O-methoxyethyl modification. The internucleoside linkagesthroughout each gapmer are phosphorothioate linkages. All cytosineresidues throughout each oligonucleotide are 5-methylcytosines. “Startsite” indicates the 5′-most nucleoside to which the gapmer is targetedin the human gene sequence. “Stop site” indicates the 3′-most nucleosideto which the gapmer is targeted in the human gene sequence. Each gapmerlisted in the tables below is targeted to either the human PKK mRNA,designated herein as SEQ ID NO: 1 or the human PKK genomic sequence,designated herein as SEQ ID NO: 10. ‘n/a’ indicates that the antisenseoligonucleotide does not target that particular gene sequence.

Cultured HepaRG™ cells at a density of 20,000 cells per well weretransfected using electroporation with 1,000 nM antisenseoligonucleotide. After a treatment period of approximately 24 hours, RNAwas isolated from the cells and PKK mRNA levels were measured byquantitative real-time PCR. Human primer probe set RTS3454 was used tomeasure mRNA levels. ISIS 531231 was also included in this assay. PKKmRNA levels were adjusted according to total RNA content, as measured byRIBOGREEN®. The antisense oligonucleotides were tested in a series ofexperiments that had similar culture conditions. The results for eachexperiment are presented in separate tables shown below. Results arepresented as percent inhibition of PKK, relative to untreated controlcells.

TABLE 19 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 SEQ ISISStart Stop % Start Stop ID NO Site Site Sequence Chemistry inhibitionSite Site NO 547747 n/a n/a TCACTGTACTAGTTTC eekd₁₀kke 95 14746 147611267 14817 14832 14888 14903 14947 14962 15007 15022 15079 15094 1522215237 15294 15309 15353 15368 15413 15428 15485 15500 15557 15572 1561515630 15687 15702 15817 15832 15889 15904 15947 15962 548074 1642 1657CCTTTCTCCTTCGAGA eekd₁₀kke 0 31948 31963 1317 548075 1643 1658ACCTTTCTCCTTCGAG eekd₁₀kke 0 31949 31964 1318 548076 1644 1659CACCTTTCTCCTTCGA eekd₁₀kke 26 n/a n/a 1319 548077 1691 1706ATTTGTTACCAAAGGA eekd₁₀kke 51 33135 33150 1320 548078 1696 1711TCTTCATTTGTTACCA eekd₁₀kke 36 33140 33155 1321 548079 1762 1777CCTTCTTTATAGCCAG eekd₁₀kke 39 33206 33221 1322 548080 1763 1778CCCTTCTTTATAGCCA eekd₁₀kke 0 33207 33222 1323 548081 1764 1779CCCCTTCTTTATAGCC eekd₁₀kke 64 33208 33223 1324 548082 1776 1791AAGCATCTTTTCCCCC eekd₁₀kke 42 33220 33235 1325 548083 1800 1815AGGGACCACCTGAATC eekd₁₀kke 0 33899 33914 1326 548084 1801 1816AAGGGACCACCTGAAT eekd₁₀kke 0 33900 33915 1327 548085 1802 1817TAAGGGACCACCTGAA eekd₁₀kke 8 33901 33916 1328 548086 1803 1818CTAAGGGACCACCTGA eekd₁₀kke 36 33902 33917 1329 548087 1804 1819ACTAAGGGACCACCTG eekd₁₀kke 24 33903 33918 1330 548088 1805 1820AACTAAGGGACCACCT eekd₁₀kke 27 33904 33919 1331 548089 1806 1821AAACTAAGGGACCACC eekd₁₀kke 34 33905 33920 1332 548090 1807 1822CAAACTAAGGGACCAC eekd₁₀kke 46 33906 33921 1333 548091 1809 1824TGCAAACTAAGGGACC eekd₁₀kke 62 33908 33923 1334 548092 1810 1825TTGCAAACTAAGGGAC eekd₁₀kke 30 33909 33924 1335 548093 1811 1826TTTGCAAACTAAGGGA eekd₁₀kke 0 33910 33925 1336 548094 1812 1827GTTTGCAAACTAAGGG eekd₁₀kke 74 33911 33926 1337 548095 1813 1828TGTTTGCAAACTAAGG eekd₁₀kke 35 33912 33927 1338 548096 1814 1829GTGTTTGCAAACTAAG eekd₁₀kke 23 33913 33928 1339 548097 1876 1891TGCTCCCTGCGGGCAC eekd₁₀kke 2 33975 33990 1340 548098 1887 1902AGACACCAGGTTGCTC eekd₁₀kke 0 33986 34001 1341 548099 1904 1919CTCAGCGACTTTGGTG eekd₁₀kke 55 34003 34018 1342 548100 1905 1920ACTCAGCGACTTTGGT eekd₁₀kke 25 34004 34019 1343 548101 1906 1921TACTCAGCGACTTTGG eekd₁₀kke 47 34005 34020 1344 548102 1907 1922GTACTCAGCGACTTTG eekd₁₀kke 58 34006 34021 1345 548103 1908 1923TGTACTCAGCGACTTT eekd₁₀kke 66 34007 34022 1346 548104 1909 1924ATGTACTCAGCGACTT eekd₁₀kke 59 34008 34023 1347 548105 1910 1925CATGTACTCAGCGACT eekd₁₀kke 49 34009 34024 1348 548106 1911 1926CCATGTACTCAGCGAC eekd₁₀kke 79 34010 34025 1349 548107 1912 1927TCCATGTACTCAGCGA eekd₁₀kke 76 34011 34026 1350 548108 1953 1968GAGCTTTTCCATCACT eekd₁₀kke 61 34052 34067 1351 548109 1959 1974GCATCTGAGCTTTTCC eekd₁₀kke 77 34058 34073 1352 548110 1960 1975TGCATCTGAGCTTTTC eekd₁₀kke 62 34059 34074 1353 548111 1963 1978GACTGCATCTGAGCTT eekd₁₀kke 53 34062 34077 1354 548112 1965 1980GTGACTGCATCTGAGC eekd₁₀kke 23 34064 34079 1355 548113 1966 1981GGTGACTGCATCTGAG eekd₁₀kke 56 34065 34080 1356 548114 1967 1982TGGTGACTGCATCTGA eekd₁₀kke 70 34066 34081 1357 548115 1972 1987CATGCTGGTGACTGCA eekd₁₀kke 76 34071 34086 1358 548116 1973 1988TCATGCTGGTGACTGC eekd₁₀kke 3 34072 34087 1359 548117 1974 1989CTCATGCTGGTGACTG eekd₁₀kke 73 34073 34088 1360 548118 1975 1990TCTCATGCTGGTGACT eekd₁₀kke 47 34074 34089 1361 548119 1984 1999TGGACTGCTTCTCATG eekd₁₀kke 25 34083 34098 1362 548121 1986 2001TCTGGACTGCTTCTCA eekd₁₀kke 64 34085 34100 1363 548122 1987 2002CTCTGGACTGCTTCTC eekd₁₀kke 55 34086 34101 1364 548123 1990 2005AGACTCTGGACTGCTT eekd₁₀kke 49 34089 34104 1365 548124 1991 2006TAGACTCTGGACTGCT eekd₁₀kke 51 34090 34105 1366 548125 1992 2007CTAGACTCTGGACTGC eekd₁₀kke 89 34091 34106 1367 548126 1995 2010TGCCTAGACTCTGGAC eekd₁₀kke 19 34094 34109 1368 548127 1996 2011TTGCCTAGACTCTGGA eekd₁₀kke 60 34095 34110 1369 548128 1997 2012ATTGCCTAGACTCTGG eekd₁₀kke 55 34096 34111 1370 548129 2022 2037TTTGACTTGAACTCAG eekd₁₀kke 35 34121 34136 1371 548130 2023 2038ATTTGACTTGAACTCA eekd₁₀kke 27 34122 34137 1372 548131 2024 2039AATTTGACTTGAACTC eekd₁₀kke 45 34123 34138 1373 548132 2025 2040GAATTTGACTTGAACT eekd₁₀kke 0 34124 34139 1374 548133 2026 2041AGAATTTGACTTGAAC eekd₁₀kke 23 34125 34140 1375 548134 2027 2042CAGAATTTGACTTGAA eekd₁₀kke 17 34126 34141 1376 548135 2028 2043TCAGAATTTGACTTGA eekd₁₀kke 46 34127 34142 1377 548136 2031 2046GGCTCAGAATTTGACT eekd₁₀kke 39 34130 34145 1378 548137 2032 2047AGGCTCAGAATTTGAC eekd₁₀kke 62 34131 34146 1379 548138 2036 2051CCCCAGGCTCAGAATT eekd₁₀kke 52 34135 34150 1380 548139 2047 2062AGATGAGGACCCCCCA eekd₁₀kke 56 34146 34161 1381 548140 2048 2063CAGATGAGGACCCCCC eekd₁₀kke 74 34147 34162 1382 548141 2049 2064GCAGATGAGGACCCCC eekd₁₀kke 66 34148 34163 1383 548142 2063 2078ACTCTCCATGCTTTGC eekd₁₀kke 44 34162 34177 1384 548143 2064 2079CACTCTCCATGCTTTG eekd₁₀kke 39 34163 34178 1385 548144 2068 2083ATGCCACTCTCCATGC eekd₁₀kke 52 34167 34182 1386 548145 2079 2094ATGCAAAGAAGATGCC eekd₁₀kke 63 34178 34193 1387 548146 2088 2103GTCCTTAGGATGCAAA eekd₁₀kke 68 34187 34202 1388 548147 2089 2104CGTCCTTAGGATGCAA eekd₁₀kke 81 34188 34203 1389 548148 2114 2129GCAGCTCTGAGTGCAC eekd₁₀kke 66 34213 34228 1390 548149 2127 2142GACATTGTCCTCAGCA eekd₁₀kke 39 34226 34241 1391 548150 2129 2144CAGACATTGTCCTCAG eekd₁₀kke 60 34228 34243 1392

TABLE 20 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 SEQ ISISStart Stop % Start Stop ID NO Site Site Sequence Chemistry inhibitionSite Site NO 547747 n/a n/a TCACTGTACTAGTTTC eekd₁₀kke 84 14746 147611267 14817 14832 14888 14903 14947 14962 15007 15022 15079 15094 1522215237 15294 15309 15353 15368 15413 15428 15485 15500 15557 15572 1561515630 15687 15702 15817 15832 15889 15904 15947 15962 547843 384 399CACTTATTTGATGACC eekd₁₀kke 83 9918 9933 1393 547844 385 400GCACTTATTTGATGAC eekd₁₀kke 13 n/a n/a 1394 547845 394 409CGATGGCAAGCACTTA eekd₁₀kke 0 n/a n/a 1395 547846 395 410TCGATGGCAAGCACTT eekd₁₀kke 0 n/a n/a 1396 547847 396 411CTCGATGGCAAGCACT eekd₁₀kke 46 n/a n/a 1397 547848 400 415ATGTCTCGATGGCAAG eekd₁₀kke 93 12656 12671 1398 547849 401 416AATGTCTCGATGGCAA eekd₁₀kke 79 12657 12672 1399 547850 402 417AAATGTCTCGATGGCA eekd₁₀kke 51 12658 12673 1400 547851 403 418TAAATGTCTCGATGGC eekd₁₀kke 93 12659 12674 1401 547852 404 419ATAAATGTCTCGATGG eekd₁₀kke 67 12660 12675 1402 547853 405 420TATAAATGTCTCGATG eekd₁₀kke 0 12661 12676 1403 547854 416 431ATCAACTCCTTTATAA eekd₁₀kke 10 12672 12687 1404 547855 417 432TATCAACTCCTTTATA eekd₁₀kke 59 12673 12688 1405 547856 419 434CATATCAACTCCTTTA eekd₁₀kke 93 12675 12690 1406 547858 423 438CTCTCATATCAACTCC eekd₁₀kke 82 12679 12694 1407 547859 424 439CCTCTCATATCAACTC eekd₁₀kke 77 12680 12695 1408 547860 425 440TCCTCTCATATCAACT eekd₁₀kke 71 12681 12696 1409 547861 427 442ACTCCTCTCATATCAA eekd₁₀kke 0 12683 12698 1410 547862 428 443GACTCCTCTCATATCA eekd₁₀kke 22 12684 12699 1411 547863 429 444TGACTCCTCTCATATC eekd₁₀kke 73 12685 12700 1412 547864 430 445TTGACTCCTCTCATAT eekd₁₀kke 53 12686 12701 1413 547865 434 449AAAATTGACTCCTCTC eekd₁₀kke 3 12690 12705 1414 547866 436 451TTAAAATTGACTCCTC eekd₁₀kke 46 12692 12707 1415 547867 447 462CCTTAGACACATTAAA eekd₁₀kke 34 12703 12718 1416 547868 448 463ACCTTAGACACATTAA eekd₁₀kke 47 12704 12719 1417 547869 449 464AACCTTAGACACATTA eekd₁₀kke 45 12705 12720 1418 547870 451 466CTAACCTTAGACACAT eekd₁₀kke 89 12707 12722 1419 547871 452 467GCTAACCTTAGACACA eekd₁₀kke 96 12708 12723 1420 547872 453 468TGCTAACCTTAGACAC eekd₁₀kke 85 12709 12724 1421 547873 454 469CTGCTAACCTTAGACA eekd₁₀kke 77 12710 12725 1422 547874 455 470ACTGCTAACCTTAGAC eekd₁₀kke 70 12711 12726 1423 547875 456 471CACTGCTAACCTTAGA eekd₁₀kke 73 12712 12727 1424 547876 457 472ACACTGCTAACCTTAG eekd₁₀kke 78 12713 12728 1425 547877 458 473AACACTGCTAACCTTA eekd₁₀kke 81 12714 12729 1426 547879 460 475TCAACACTGCTAACCT eekd₁₀kke 69 12716 12731 1427 547880 461 476TTCAACACTGCTAACC eekd₁₀kke 69 12717 12732 1428 547881 465 480ATTCTTCAACACTGCT eekd₁₀kke 0 12721 12736 1429 547882 500 515CTGGCAGCGAATGTTA eekd₁₀kke 91 12756 12771 1430 547883 501 516ACTGGCAGCGAATGTT eekd₁₀kke 99 12757 12772 1431 547884 518 533CGTGGCATATGAAAAA eekd₁₀kke 87 12774 12789 1432 547885 539 554CTCTGCCTTGTGAAAT eekd₁₀kke 45 12795 12810 1433 547886 544 559CGGTACTCTGCCTTGT eekd₁₀kke 97 12800 12815 1434 547889 547 562TTCCGGTACTCTGCCT eekd₁₀kke 91 n/a n/a 1435 547890 550 565TTGTTCCGGTACTCTG eekd₁₀kke 97 n/a n/a 1436 547891 551 566ATTGTTCCGGTACTCT eekd₁₀kke 84 n/a n/a 1437 547892 553 568CAATTGTTCCGGTACT eekd₁₀kke 29 n/a n/a 1438 547893 554 569GCAATTGTTCCGGTAC eekd₁₀kke 81 n/a n/a 1439 547894 555 570GGCAATTGTTCCGGTA eekd₁₀kke 92 n/a n/a 1440 547898 563 578CTTTAATAGGCAATTG eekd₁₀kke 0 14134 14149 1441 547899 566 581GTACTTTAATAGGCAA eekd₁₀kke 49 14137 14152 1442 547900 567 582TGTACTTTAATAGGCA eekd₁₀kke 93 14138 14153 1443 547901 568 583CTGTACTTTAATAGGC eekd₁₀kke 77 14139 14154 1444 547902 569 584ACTGTACTTTAATAGG eekd₁₀kke 20 14140 14155 1445 547903 604 619CTCAGCACCTTTATAG eekd₁₀kke 62 14175 14190 1446 547904 605 620ACTCAGCACCTTTATA eekd₁₀kke 56 14176 14191 1447 547905 606 621TACTCAGCACCTTTAT eekd₁₀kke 20 14177 14192 1448 547906 607 622TTACTCAGCACCTTTA eekd₁₀kke 59 14178 14193 1449 547907 652 667ATTTCTGAAAGGGCAC eekd₁₀kke 27 14223 14238 1450 547908 654 669CAATTTCTGAAAGGGC eekd₁₀kke 94 14225 14240 1451 547909 655 670CCAATTTCTGAAAGGG eekd₁₀kke 82 14226 14241 1452 547910 656 671ACCAATTTCTGAAAGG eekd₁₀kke 26 14227 14242 1453 547911 661 676TGGCAACCAATTTCTG eekd₁₀kke 0 n/a n/a 1454 547912 701 716ATCCACATCTGAGAAC eekd₁₀kke 23 26149 26164 1455 547913 706 721GCAACATCCACATCTG eekd₁₀kke 71 26154 26169 1456 547914 707 722GGCAACATCCACATCT eekd₁₀kke 74 26155 26170 1457 547915 708 723TGGCAACATCCACATC eekd₁₀kke 0 26156 26171 1458 547916 710 725CCTGGCAACATCCACA eekd₁₀kke 70 26158 26173 1459 547917 712 727ACCCTGGCAACATCCA eekd₁₀kke 33 26160 26175 1460 547918 713 728AACCCTGGCAACATCC eekd₁₀kke 1 26161 26176 1461 547919 714 729GAACCCTGGCAACATC eekd₁₀kke 41 26162 26177 1462

TABLE 21 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 ISISStart Stop % Start Stop SEQ NO Site Site Sequence Chemistry inhibitionSite Site ID NO 531231 n/a n/a TATCACTGTACTAGTTTCCT eeeeed₁₀eeeee 6214744 14763 334 14815 14834 14886 14905 14945 14964 15005 15024 1507715096 15220 15239 15292 15311 15351 15370 15411 15430 15483 15502 1555515574 15613 15632 15685 15704 15815 15834 15887 15906 15945 15964 547747n/a n/a TCACTGTACTAGTTTC eekd₁₀kke 88 14746 14761 1267 14817 14832 1488814903 14947 14962 15007 15022 15079 15094 15222 15237 15294 15309 1535315368 15413 15428 15485 15500 15557 15572 15615 15630 15687 15702 1581715832 15889 15904 15947 15962 547751 7 22 TGAACGGTCTTCAAGC eekd₁₀kke 03399 3414 1463 547753 8 23 ATGAACGGTCTTCAAG eekd₁₀kke 3 3400 3415 1464547755 13 28 TAAAAATGAACGGTCT eekd₁₀kke 0 3405 3420 1465 547757 28 43GAGTCTCTTGTCACTT eekd₁₀kke 69 3420 3435 1466 547759 29 44TGAGTCTCTTGTCACT eekd₁₀kke 73 3421 3436 1467 547763 31 46GGTGAGTCTCTTGTCA eekd₁₀kke 66 3423 3438 1468 547765 32 47AGGTGAGTCTCTTGTC eekd₁₀kke 20 3424 3439 1469 547767 35 50TGGAGGTGAGTCTCTT eekd₁₀kke 74 3427 3442 1470 547769 36 51TTGGAGGTGAGTCTCT eekd₁₀kke 81 3428 3443 1471 547771 37 52CTTGGAGGTGAGTCTC eekd₁₀kke 60 3429 3444 1472 547773 38 53TCTTGGAGGTGAGTCT eekd₁₀kke 47 3430 3445 1473 547777 43 58TTGCTTCTTGGAGGTG eekd₁₀kke 69 3435 3450 1474 547779 44 59ATTGCTTCTTGGAGGT eekd₁₀kke 41 3436 3451 1475 547781 46 61CAATTGCTTCTTGGAG eekd₁₀kke 49 3438 3453 1476 547783 48 63CACAATTGCTTCTTGG eekd₁₀kke 48 3440 3455 1477 547784 72 87GCTTGAATAAAATCAT eekd₁₀kke 46 4071 4086 1478 547785 79 94GTTGCTTGCTTGAATA eekd₁₀kke 48 4078 4093 1479 547786 80 95AGTTGCTTGCTTGAAT eekd₁₀kke 44 4079 4094 1480 547787 81 96AAGTTGCTTGCTTGAA eekd₁₀kke 22 4080 4095 1481 547788 82 97TAAGTTGCTTGCTTGA eekd₁₀kke 49 4081 4096 1482 547789 86 101GAAATAAGTTGCTTGC eekd₁₀kke 20 4085 4100 1483 547790 87 102TGAAATAAGTTGCTTG eekd₁₀kke 23 4086 4101 1484 547791 106 121ACTGTAGCAAACAAGG eekd₁₀kke 49 4105 4120 1485 547792 116 131TCCACAGGAAACTGTA eekd₁₀kke 31 n/a n/a 1486 547793 117 132ATCCACAGGAAACTGT eekd₁₀kke 16 n/a n/a 1487 547794 136 151TCATAGAGTTGAGTCA eekd₁₀kke 49 8008 8023 1488 547795 155 170ACCTCTGAAGAAGGCG eekd₁₀kke 66 8027 8042 1489 547796 161 176ATCCCCACCTCTGAAG eekd₁₀kke 35 8033 8048 1490 547797 167 182AGCTACATCCCCACCT eekd₁₀kke 33 8039 8054 1491 547799 169 184GAAGCTACATCCCCAC eekd₁₀kke 41 8041 8056 1492 547800 174 189ACATGGAAGCTACATC eekd₁₀kke 20 8046 8061 1493 547801 175 190TACATGGAAGCTACAT eekd₁₀kke 11 8047 8062 1494 547802 176 191GTACATGGAAGCTACA eekd₁₀kke 41 8048 8063 1495 547803 177 192TGTACATGGAAGCTAC eekd₁₀kke 0 8049 8064 1496 547804 178 193GTGTACATGGAAGCTA eekd₁₀kke 22 8050 8065 1497 547805 180 195GGGTGTACATGGAAGC eekd₁₀kke 54 8052 8067 1498 547807 197 212GCAGTATTGGGCATTT eekd₁₀kke 75 8069 8084 1499 547808 203 218CATCTGGCAGTATTGG eekd₁₀kke 56 8075 8090 1500 547809 204 219TCATCTGGCAGTATTG eekd₁₀kke 33 8076 8091 1501 547810 206 221CCTCATCTGGCAGTAT eekd₁₀kke 60 8078 8093 1502 547811 207 222ACCTCATCTGGCAGTA eekd₁₀kke 49 8079 8094 1503 547812 211 226GTGCACCTCATCTGGC eekd₁₀kke 51 8083 8098 1504 547813 219 234GGTGGAATGTGCACCT eekd₁₀kke 34 8091 8106 1505 547814 220 235GGGTGGAATGTGCACC eekd₁₀kke 60 8092 8107 1506 547815 255 270AACTTGCTGGAAGAAA eekd₁₀kke 3 8127 8142 1507 547816 256 271GAACTTGCTGGAAGAA eekd₁₀kke 45 8128 8143 1508 547817 257 272TGAACTTGCTGGAAGA eekd₁₀kke 18 8129 8144 1509 547818 260 275GATTGAACTTGCTGGA eekd₁₀kke 4 8132 8147 1510 547819 264 279CATTGATTGAACTTGC eekd₁₀kke 11 8136 8151 1511 547820 265 280TCATTGATTGAACTTG eekd₁₀kke 0 8137 8152 1512 547821 282 297CAAACCTTTTCTCCAT eekd₁₀kke 44 n/a n/a 1513 547822 287 302GCAACCAAACCTTTTC eekd₁₀kke 71 n/a n/a 1514 547823 288 303AGCAACCAAACCTTTT eekd₁₀kke 51 n/a n/a 1515 547824 331 346CGATGTACTTTTGGCA eekd₁₀kke 82 9865 9880 1516 547825 332 347TCGATGTACTTTTGGC eekd₁₀kke 59 9866 9881 1517 547826 333 348TTCGATGTACTTTTGG eekd₁₀kke 31 9867 9882 1518 547827 334 349GTTCGATGTACTTTTG eekd₁₀kke 47 9868 9883 1519 547828 337 352CCTGTTCGATGTACTT eekd₁₀kke 63 9871 9886 1520 547829 338 353ACCTGTTCGATGTACT eekd₁₀kke 59 9872 9887 1521 547830 340 355GCACCTGTTCGATGTA eekd₁₀kke 74 9874 9889 1522 547831 342 357CTGCACCTGTTCGATG eekd₁₀kke 49 9876 9891 1523 547832 343 358ACTGCACCTGTTCGAT eekd₁₀kke 59 9877 9892 1524 547833 344 359AACTGCACCTGTTCGA eekd₁₀kke 40 9878 9893 1525 547834 345 360AAACTGCACCTGTTCG eekd₁₀kke 63 9879 9894 1526 547835 349 364CCAGAAACTGCACCTG eekd₁₀kke 81 9883 9898 1527 547836 350 365TCCAGAAACTGCACCT eekd₁₀kke 50 9884 9899 1528 547837 352 367TGTCCAGAAACTGCAC eekd₁₀kke 51 9886 9901 1529 547838 362 377CTTCAAGGAATGTCCA eekd₁₀kke 45 9896 9911 1530 547839 363 378GCTTCAAGGAATGTCC eekd₁₀kke 35 9897 9912 1531 547840 365 380TTGCTTCAAGGAATGT eekd₁₀kke 36 9899 9914 1532 547841 369 384CACATTGCTTCAAGGA eekd₁₀kke 42 9903 9918 1533 547842 375 390GATGACCACATTGCTT eekd₁₀kke 10 9909 9924 1534

TABLE 22 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 ISISStart Stop % Start Stop SEQ ID NO Site Site Sequence Chemistryinhibition Site Site NO 531231 n/a n/a TATCACTGTACTAGTTTCCTeeeeed₁₀eeeee 75 14744 14763 334 14815 14834 14886 14905 14945 1496415005 15024 15077 15096 15220 15239 15292 15311 15351 15370 15411 1543015483 15502 15555 15574 15613 15632 15685 15704 15815 15834 15887 1590615945 15964 547747 n/a n/a TCACTGTACTAGTTTC eekd₁₀kke 91 14746 147611267 14817 14832 14888 14903 14947 14962 15007 15022 15079 15094 1522215237 15294 15309 15353 15368 15413 15428 15485 15500 15557 15572 1561515630 15687 15702 15817 15832 15889 15904 15947 15962 547843 384 399CACTTATTTGATGACC eekd₁₀kke 83 9918 9933 1393 547844 385 400GCACTTATTTGATGAC eekd₁₀kke 76 n/a n/a 1394 547845 394 409CGATGGCAAGCACTTA eekd₁₀kke 64 n/a n/a 1395 547846 395 410TCGATGGCAAGCACTT eekd₁₀kke 42 n/a n/a 1396 547847 396 411CTCGATGGCAAGCACT eekd₁₀kke 72 n/a n/a 1397 547848 400 415ATGTCTCGATGGCAAG eekd₁₀kke 79 12656 12671 1398 547849 401 416AATGTCTCGATGGCAA eekd₁₀kke 90 12657 12672 1399 547850 402 417AAATGTCTCGATGGCA eekd₁₀kke 80 12658 12673 1400 547851 403 418TAAATGTCTCGATGGC eekd₁₀kke 84 12659 12674 1401 547852 404 419ATAAATGTCTCGATGG eekd₁₀kke 66 12660 12675 1402 547853 405 420TATAAATGTCTCGATG eekd₁₀kke 30 12661 12676 1403 547854 416 431ATCAACTCCTTTATAA eekd₁₀kke 9 12672 12687 1404 547855 417 432TATCAACTCCTTTATA eekd₁₀kke 38 12673 12688 1405 547856 419 434CATATCAACTCCTTTA eekd₁₀kke 51 12675 12690 1406 547857 421 436CTCATATCAACTCCTT eekd₁₀kke 84 12677 12692 1535 547858 423 438CTCTCATATCAACTCC eekd₁₀kke 76 12679 12694 1407 547859 424 439CCTCTCATATCAACTC eekd₁₀kke 88 12680 12695 1408 547860 425 440TCCTCTCATATCAACT eekd₁₀kke 70 12681 12696 1409 547861 427 442ACTCCTCTCATATCAA eekd₁₀kke 57 12683 12698 1410 547862 428 443GACTCCTCTCATATCA eekd₁₀kke 88 12684 12699 1411 547863 429 444TGACTCCTCTCATATC eekd₁₀kke 77 12685 12700 1412 547864 430 445TTGACTCCTCTCATAT eekd₁₀kke 73 12686 12701 1413 547865 434 449AAAATTGACTCCTCTC eekd₁₀kke 61 12690 12705 1414 547866 436 451TTAAAATTGACTCCTC eekd₁₀kke 40 12692 12707 1415 547867 447 462CCTTAGACACATTAAA eekd₁₀kke 53 12703 12718 1416 547868 448 463ACCTTAGACACATTAA eekd₁₀kke 71 12704 12719 1417 547869 449 464AACCTTAGACACATTA eekd₁₀kke 77 12705 12720 1418 547870 451 466CTAACCTTAGACACAT eekd₁₀kke 83 12707 12722 1419 547871 452 467GCTAACCTTAGACACA eekd₁₀kke 77 12708 12723 1420 547872 453 468TGCTAACCTTAGACAC eekd₁₀kke 73 12709 12724 1421 547873 454 469CTGCTAACCTTAGACA eekd₁₀kke 82 12710 12725 1422 547874 455 470ACTGCTAACCTTAGAC eekd₁₀kke 60 12711 12726 1423 547875 456 471CACTGCTAACCTTAGA eekd₁₀kke 57 12712 12727 1424 547876 457 472ACACTGCTAACCTTAG eekd₁₀kke 59 12713 12728 1425 547877 458 473AACACTGCTAACCTTA eekd₁₀kke 93 12714 12729 1426 547878 459 474CAACACTGCTAACCTT eekd₁₀kke 62 12715 12730 1536 547879 460 475TCAACACTGCTAACCT eekd₁₀kke 65 12716 12731 1427 547880 461 476TTCAACACTGCTAACC eekd₁₀kke 59 12717 12732 1428 547881 465 480ATTCTTCAACACTGCT eekd₁₀kke 50 12721 12736 1429 547882 500 515CTGGCAGCGAATGTTA eekd₁₀kke 96 12756 12771 1430 547883 501 516ACTGGCAGCGAATGTT eekd₁₀kke 0 12757 12772 1431 547884 518 533CGTGGCATATGAAAAA eekd₁₀kke 49 12774 12789 1432 547885 539 554CTCTGCCTTGTGAAAT eekd₁₀kke 57 12795 12810 1433 547886 544 559CGGTACTCTGCCTTGT eekd₁₀kke 89 12800 12815 1434 547887 545 560CCGGTACTCTGCCTTG eekd₁₀kke 99 12801 12816 1537 547888 546 561TCCGGTACTCTGCCTT eekd₁₀kke 99 n/a n/a 1538 547889 547 562TTCCGGTACTCTGCCT eekd₁₀kke 97 n/a n/a 1435 547890 550 565TTGTTCCGGTACTCTG eekd₁₀kke 90 n/a n/a 1436 547891 551 566ATTGTTCCGGTACTCT eekd₁₀kke 88 n/a n/a 1437 547892 553 568CAATTGTTCCGGTACT eekd₁₀kke 28 n/a n/a 1438 547893 554 569GCAATTGTTCCGGTAC eekd₁₀kke 80 n/a n/a 1439 547894 555 570GGCAATTGTTCCGGTA eekd₁₀kke 91 n/a n/a 1440 547895 556 571AGGCAATTGTTCCGGT eekd₁₀kke 94 n/a n/a 1539 547896 557 572TAGGCAATTGTTCCGG eekd₁₀kke 95 n/a n/a 1540 547897 558 573ATAGGCAATTGTTCCG eekd₁₀kke 82 n/a n/a 1541 547898 563 578CTTTAATAGGCAATTG eekd₁₀kke 28 14134 14149 1441 547899 566 581GTACTTTAATAGGCAA eekd₁₀kke 68 14137 14152 1442 547900 567 582TGTACTTTAATAGGCA eekd₁₀kke 68 14138 14153 1443 547901 568 583CTGTACTTTAATAGGC eekd₁₀kke 85 14139 14154 1444 547902 569 584ACTGTACTTTAATAGG eekd₁₀kke 33 14140 14155 1445 547903 604 619CTCAGCACCTTTATAG eekd₁₀kke 6 14175 14190 1446 547904 605 620ACTCAGCACCTTTATA eekd₁₀kke 41 14176 14191 1447 547905 606 621TACTCAGCACCTTTAT eekd₁₀kke 59 14177 14192 1448 547906 607 622TTACTCAGCACCTTTA eekd₁₀kke 70 14178 14193 1449 547907 652 667ATTTCTGAAAGGGCAC eekd₁₀kke 27 14223 14238 1450 547908 654 669CAATTTCTGAAAGGGC eekd₁₀kke 71 14225 14240 1451 547909 655 670CCAATTTCTGAAAGGG eekd₁₀kke 51 14226 14241 1452 547910 656 671ACCAATTTCTGAAAGG eekd₁₀kke 34 14227 14242 1453 547911 661 676TGGCAACCAATTTCTG eekd₁₀kke 15 n/a n/a 1454 547912 701 716ATCCACATCTGAGAAC eekd₁₀kke 53 26149 26164 1455 547913 706 721GCAACATCCACATCTG eekd₁₀kke 61 26154 26169 1456 547914 707 722GGCAACATCCACATCT eekd₁₀kke 63 26155 26170 1457 547915 708 723TGGCAACATCCACATC eekd₁₀kke 62 26156 26171 1458 547916 710 725CCTGGCAACATCCACA eekd₁₀kke 56 26158 26173 1459 547917 712 727ACCCTGGCAACATCCA eekd₁₀kke 54 26160 26175 1460 547918 713 728AACCCTGGCAACATCC eekd₁₀kke 65 26161 26176 1461 547919 714 729GAACCCTGGCAACATC eekd₁₀kke 73 26162 26177 1462

TABLE 23 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 ISISStart Stop % Start Stop SEQ NO Site Site Sequence Chemistry inhibitionSite Site ID NO 531231 n/a n/a TATCACTGTACTAGTTTCCT eeeeed₁₀eeeee 1614744 14763 334 14815 14834 14886 14905 14945 14964 15005 15024 1507715096 15220 15239 15292 15311 15351 15370 15411 15430 15483 15502 1555515574 15613 15632 15685 15704 15815 15834 15887 15906 15945 15964 547747n/a n/a TCACTGTACTAGTTTC eekd₁₀kke 83 14746 14761 1267 14817 14832 1488814903 14947 14962 15007 15022 15079 15094 15222 15237 15294 15309 1535315368 15413 15428 15485 15500 15557 15572 15615 15630 15687 15702 1581715832 15889 15904 15947 15962 547920 716 731 GAGAACCCTGGCAACA eekd₁₀kke52 26164 26179 1542 547921 717 732 TGAGAACCCTGGCAAC eekd₁₀kke 43 2616526180 1543 547922 722 737 TGGAGTGAGAACCCTG eekd₁₀kke 79 26170 26185 1544547923 725 740 ATCTGGAGTGAGAACC eekd₁₀kke 68 26173 26188 1545 547924 742757 GTCCGACACACAAAAG eekd₁₀kke 53 26190 26205 1546 547925 743 758GGTCCGACACACAAAA eekd₁₀kke 16 26191 26206 1547 547927 745 760ATGGTCCGACACACAA eekd₁₀kke 79 26193 26208 1548 547928 746 761GATGGTCCGACACACA eekd₁₀kke 70 26194 26209 1549 547929 747 762AGATGGTCCGACACAC eekd₁₀kke 65 26195 26210 1550 547930 757 772TGATAGGTGCAGATGG eekd₁₀kke 48 26205 26220 1551 547931 758 773GTGATAGGTGCAGATG eekd₁₀kke 58 26206 26221 1552 547932 804 819CGATTTTCCATACATT eekd₁₀kke 33 26252 26267 1553 547933 805 820TCGATTTTCCATACAT eekd₁₀kke 44 26253 26268 1554 547934 806 821CTCGATTTTCCATACA eekd₁₀kke 38 26254 26269 1555 547935 807 822ACTCGATTTTCCATAC eekd₁₀kke 27 26255 26270 1556 547936 808 823GACTCGATTTTCCATA eekd₁₀kke 44 26256 26271 1557 547937 811 826TGTGACTCGATTTTCC eekd₁₀kke 56 26259 26274 1558 547938 812 827TTGTGACTCGATTTTC eekd₁₀kke 56 26260 26275 1559 547939 813 828TTTGTGACTCGATTTT eekd₁₀kke 70 26261 26276 1560 547940 817 832TTTCTTTGTGACTCGA eekd₁₀kke 71 n/a n/a 1561 547941 852 867GTGTGCCACTTTCAGA eekd₁₀kke 66 27116 27131 1562 547942 853 868GGTGTGCCACTTTCAG eekd₁₀kke 85 27117 27132 1563 547943 854 869TGGTGTGCCACTTTCA eekd₁₀kke 83 27118 27133 1564 547944 857 872ACTTGGTGTGCCACTT eekd₁₀kke 54 27121 27136 1565 547945 858 873AACTTGGTGTGCCACT eekd₁₀kke 62 27122 27137 1566 547946 859 874GAACTTGGTGTGCCAC eekd₁₀kke 81 27123 27138 1567 547947 860 875GGAACTTGGTGTGCCA eekd₁₀kke 80 27124 27139 1568 547948 861 876AGGAACTTGGTGTGCC eekd₁₀kke 77 27125 27140 1569 547949 880 895GTGTTTTCTTGAGGAG eekd₁₀kke 6 27144 27159 1570 547950 881 896GGTGTTTTCTTGAGGA eekd₁₀kke 49 27145 27160 1571 547951 887 902AGATATGGTGTTTTCT eekd₁₀kke 25 27151 27166 1572 547952 888 903CAGATATGGTGTTTTC eekd₁₀kke 46 27152 27167 1573 547953 895 910CTATATCCAGATATGG eekd₁₀kke 16 27159 27174 1574 547954 902 917TAAAAGGCTATATCCA eekd₁₀kke 36 27166 27181 1575 547956 904 919GTTAAAAGGCTATATC eekd₁₀kke 13 27168 27183 1576 547957 905 920GGTTAAAAGGCTATAT eekd₁₀kke 6 27169 27184 1577 547958 907 922CAGGTTAAAAGGCTAT eekd₁₀kke 57 27171 27186 1578 547959 908 923GCAGGTTAAAAGGCTA eekd₁₀kke 60 27172 27187 1579 547960 909 924TGCAGGTTAAAAGGCT eekd₁₀kke 40 27173 27188 1580 547961 910 925TTGCAGGTTAAAAGGC eekd₁₀kke 5 27174 27189 1581 547962 911 926TTTGCAGGTTAAAAGG eekd₁₀kke 16 27175 27190 1582 547963 927 942GTTCAGGTAAAGTTCT eekd₁₀kke 22 n/a n/a 1583 547964 928 943GGTTCAGGTAAAGTTC eekd₁₀kke 0 n/a n/a 1584 547965 929 944GGGTTCAGGTAAAGTT eekd₁₀kke 29 n/a n/a 1585 547966 930 945AGGGTTCAGGTAAAGT eekd₁₀kke 13 n/a n/a 1586 547967 933 948GGCAGGGTTCAGGTAA eekd₁₀kke 25 n/a n/a 1587 547968 940 955TTAGAATGGCAGGGTT eekd₁₀kke 37 27362 27377 1588 547969 953 968TCCCGGGTAAATTTTA eekd₁₀kke 0 27375 27390 1589 547970 954 969CTCCCGGGTAAATTTT eekd₁₀kke 42 27376 27391 1590 547972 958 973TCAACTCCCGGGTAAA eekd₁₀kke 49 27380 27395 1591 547973 961 976AAGTCAACTCCCGGGT eekd₁₀kke 62 27383 27398 1592 547974 962 977AAAGTCAACTCCCGGG eekd₁₀kke 52 27384 27399 1593 547975 963 978CAAAGTCAACTCCCGG eekd₁₀kke 44 27385 27400 1594 547976 964 979CCAAAGTCAACTCCCG eekd₁₀kke 49 27386 27401 1595 547977 967 982CCTCCAAAGTCAACTC eekd₁₀kke 57 27389 27404 1596 547978 1014 1029CTTGGCAAACATTCAC eekd₁₀kke 71 27436 27451 1597 547979 1018 1033GTCTCTTGGCAAACAT eekd₁₀kke 77 27440 27455 1598 547980 1020 1035AAGTCTCTTGGCAAAC eekd₁₀kke 54 27442 27457 1599 547981 1029 1044TCTTTGTGCAAGTCTC eekd₁₀kke 76 27451 27466 1600 547982 1034 1049AATCATCTTTGTGCAA eekd₁₀kke 54 27456 27471 1601 547983 1035 1050GAATCATCTTTGTGCA eekd₁₀kke 56 27457 27472 1602 547984 1036 1051CGAATCATCTTTGTGC eekd₁₀kke 55 27458 27473 1603 547985 1037 1052GCGAATCATCTTTGTG eekd₁₀kke 63 27459 27474 1604 547986 1039 1054CAGCGAATCATCTTTG eekd₁₀kke 63 27461 27476 1605 547987 1040 1055ACAGCGAATCATCTTT eekd₁₀kke 64 27462 27477 1606 547988 1042 1057TGACAGCGAATCATCT eekd₁₀kke 56 27464 27479 1607 547989 1043 1058CTGACAGCGAATCATC eekd₁₀kke 66 27465 27480 1608 547990 1044 1059ACTGACAGCGAATCAT eekd₁₀kke 58 27466 27481 1609 547991 1077 1092TACAGTCTTCTGGGAG eekd₁₀kke 0 27499 27514 1610 547992 1080 1095CCTTACAGTCTTCTGG eekd₁₀kke 17 27502 27517 1611 547993 1113 1128TAGATAATCTTAAGAA eekd₁₀kke 26 27634 27649 1612 547994 1120 1135CCATCCATAGATAATC eekd₁₀kke 53 27641 27656 1613 547995 1149 1164GTGTCCCATACGCAAT eekd₁₀kke 64 27670 27685 1614 547996 1150 1165TGTGTCCCATACGCAA eekd₁₀kke 65 27671 27686 1615

TABLE 24 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 SEQ ISISStart Stop % Start Stop ID NO Site Site Sequence Chemistry inhibitionSite Site NO 531231 n/a n/a TATCACTGTACTAGTTTCCT eeeeed₁₀eeeee 0 1474414763 334 14815 14834 14886 14905 14945 14964 15005 15024 15077 1509615220 15239 15292 15311 15351 15370 15411 15430 15483 15502 15555 1557415613 15632 15685 15704 15815 15834 15887 15906 15945 15964 547747 n/an/a TCACTGTACTAGTTTC eekd₁₀kke 80 14746 14761 1267 14817 14832 1488814903 14947 14962 15007 15022 15079 15094 15222 15237 15294 15309 1535315368 15413 15428 15485 15500 15557 15572 15615 15630 15687 15702 1581715832 15889 15904 15947 15962 547997 1151 1166 TTGTGTCCCATACGCAeekd₁₀kke 89 27672 27687 1616 547998 1152 1167 CTTGTGTCCCATACGCeekd₁₀kke 82 27673 27688 1617 547999 1153 1168 CCTTGTGTCCCATACGeekd₁₀kke 50 27674 27689 1618 548000 1154 1169 CCCTTGTGTCCCATACeekd₁₀kke 54 27675 27690 1619 548001 1163 1178 ACCAGAGCTCCCTTGTeekd₁₀kke 64 27684 27699 1620 548002 1164 1179 AACCAGAGCTCCCTTGeekd₁₀kke 56 27685 27700 1621 548003 1165 1180 TAACCAGAGCTCCCTTeekd₁₀kke 66 27686 27701 1622 548004 1167 1182 AGTAACCAGAGCTCCCeekd₁₀kke 80 27688 27703 1623 548005 1169 1184 AGAGTAACCAGAGCTCeekd₁₀kke 77 27690 27705 1624 548006 1172 1187 CAAAGAGTAACCAGAGeekd₁₀kke 54 27693 27708 1625 548007 1174 1189 CTCAAAGAGTAACCAGeekd₁₀kke 70 27695 27710 1626 548008 1175 1190 TCTCAAAGAGTAACCAeekd₁₀kke 71 27696 27711 1627 548009 1184 1199 GTTACACAATCTCAAAeekd₁₀kke 47 27705 27720 1628 548010 1187 1202 AGTGTTACACAATCTCeekd₁₀kke 80 27708 27723 1629 548011 1189 1204 CCAGTGTTACACAATCeekd₁₀kke 14 27710 27725 1630 548012 1192 1207 TCCCCAGTGTTACACAeekd₁₀kke 3 27713 27728 1631 548013 1193 1208 GTCCCCAGTGTTACAC eekd₁₀kke37 27714 27729 1632 548014 1194 1209 TGTCCCCAGTGTTACA eekd₁₀kke 31 2771527730 1633 548015 1195 1210 TTGTCCCCAGTGTTAC eekd₁₀kke 50 27716 277311634 548016 1248 1263 AAGAGTTTGTTCCTCC eekd₁₀kke 55 27924 27939 1635548017 1252 1267 CAAGAAGAGTTTGTTC eekd₁₀kke 3 27928 27943 1636 5480181253 1268 CCAAGAAGAGTTTGTT eekd₁₀kke 22 27929 27944 1637 548019 12551270 CCCCAAGAAGAGTTTG eekd₁₀kke 24 27931 27946 1638 548020 1256 1271TCCCCAAGAAGAGTTT eekd₁₀kke 76 27932 27947 1639 548021 1261 1276CACTCTCCCCAAGAAG eekd₁₀kke 0 27937 27952 1640 548022 1262 1277CCACTCTCCCCAAGAA eekd₁₀kke 69 27938 27953 1641 548023 1290 1305GCTTCACCTGCAGGCT eekd₁₀kke 58 27966 27981 1642 548024 1297 1312GCTGTCAGCTTCACCT eekd₁₀kke 79 27973 27988 1643 548025 1300 1315TGAGCTGTCAGCTTCA eekd₁₀kke 66 27976 27991 1644 548026 1332 1347GTCCTATGAGTGACCC eekd₁₀kke 52 28008 28023 1645 548027 1334 1349GTGTCCTATGAGTGAC eekd₁₀kke 18 28010 28025 1646 548028 1335 1350GGTGTCCTATGAGTGA eekd₁₀kke 38 28011 28026 1647 548029 1336 1351TGGTGTCCTATGAGTG eekd₁₀kke 12 28012 28027 1648 548030 1337 1352CTGGTGTCCTATGAGT eekd₁₀kke 52 28013 28028 1649 548031 1397 1412GATGCGCCAAACATCC eekd₁₀kke 73 30475 30490 1650 548032 1398 1413AGATGCGCCAAACATC eekd₁₀kke 51 30476 30491 1651 548034 1400 1415ATAGATGCGCCAAACA eekd₁₀kke 31 30478 30493 1652 548035 1404 1419CACTATAGATGCGCCA eekd₁₀kke 44 30482 30497 1653 548036 1405 1420CCACTATAGATGCGCC eekd₁₀kke 74 30483 30498 1654 548037 1427 1442AATGTCTGACAGATTT eekd₁₀kke 70 30505 30520 1655 548038 1428 1443TAATGTCTGACAGATT eekd₁₀kke 67 30506 30521 1656 548039 1445 1460GAAAGGTGTATCTTTT eekd₁₀kke 29 30523 30538 1657 548040 1449 1464GTGAGAAAGGTGTATC eekd₁₀kke 62 30527 30542 1658 548041 1450 1465TGTGAGAAAGGTGTAT eekd₁₀kke 64 30528 30543 1659 548042 1452 1467TTTGTGAGAAAGGTGT eekd₁₀kke 63 30530 30545 1660 548043 1453 1468ATTTGTGAGAAAGGTG eekd₁₀kke 76 30531 30546 1661 548044 1474 1489TGGTGAATAATAATCT eekd₁₀kke 12 30552 30567 1662 548045 1483 1498TTATAGTTTTGGTGAA eekd₁₀kke 0 30561 30576 1663 548046 1506 1521TATCATGATTCCCTTC eekd₁₀kke 84 30584 30599 1664 548047 1508 1523GATATCATGATTCCCT eekd₁₀kke 83 30586 30601 1665 548048 1509 1524CGATATCATGATTCCC eekd₁₀kke 84 30587 30602 1666 548049 1510 1525GCGATATCATGATTCC eekd₁₀kke 62 30588 30603 1667 548050 1512 1527AGGCGATATCATGATT eekd₁₀kke 37 30590 30605 1668 548051 1513 1528AAGGCGATATCATGAT eekd₁₀kke 61 30591 30606 1669 548052 1535 1550CAAAGGAGCCTGGAGT eekd₁₀kke 43 30613 30628 1670 548053 1538 1553ATTCAAAGGAGCCTGG eekd₁₀kke 36 30616 30631 1671 548054 1539 1554AATTCAAAGGAGCCTG eekd₁₀kke 45 30617 30632 1672 548055 1541 1556GTAATTCAAAGGAGCC eekd₁₀kke 78 30619 30634 1673 548056 1543 1558GTGTAATTCAAAGGAG eekd₁₀kke 40 30621 30636 1674 548057 1564 1579CATATTGGTTTTTGGA eekd₁₀kke 49 31870 31885 1675 548058 1565 1580GCATATTGGTTTTTGG eekd₁₀kke 71 31871 31886 1676 548059 1568 1583TAGGCATATTGGTTTT eekd₁₀kke 50 31874 31889 1677 548060 1588 1603CTTGTGTCACCTTTGG eekd₁₀kke 76 31894 31909 1678 548061 1589 1604GCTTGTGTCACCTTTG eekd₁₀kke 86 31895 31910 1679 548062 1598 1613ATAAATTGTGCTTGTG eekd₁₀kke 19 31904 31919 1680 548063 1600 1615GTATAAATTGTGCTTG eekd₁₀kke 35 31906 31921 1681 548064 1602 1617TGGTATAAATTGTGCT eekd₁₀kke 54 31908 31923 1682 548065 1603 1618TTGGTATAAATTGTGC eekd₁₀kke 22 31909 31924 1683 548067 1606 1621CAGTTGGTATAAATTG eekd₁₀kke 18 31912 31927 1684 548068 1609 1624CAACAGTTGGTATAAA eekd₁₀kke 0 31915 31930 1685 548069 1610 1625CCAACAGTTGGTATAA eekd₁₀kke 57 31916 31931 1686 548070 1611 1626CCCAACAGTTGGTATA eekd₁₀kke 85 31917 31932 1687 548071 1629 1644AGAAGCCCCATCCGGT eekd₁₀kke 55 31935 31950 1688 548072 1640 1655TTTCTCCTTCGAGAAG eekd₁₀kke 33 31946 31961 1689 548073 1641 1656CTTTCTCCTTCGAGAA eekd₁₀kke 24 31947 31962 1690

TABLE 25 SEQ SEQ ID ID NO: NO: SEQ ID SEQ ID 10 10 ISIS NO: 1 NO: 1 %Start Stop SEQ NO Start Site Stop Site Sequence Chemistry inhibitionSite Site ID NO 531231 n/a n/a TATCACTGTACTAGTTTCCT eeeeed₁₀eeeee 1914744 14763 334 14815 14834 14886 14905 14945 14964 15005 15024 1507715096 15220 15239 15292 15311 15351 15370 15411 15430 15483 15502 1555515574 15613 15632 15685 15704 15815 15834 15887 15906 15945 15964 547747n/a n/a TCACTGTACTAGTTTC eekd₁₀kke 66 14746 14761 1267 14817 14832 1488814903 14947 14962 15007 15022 15079 15094 15222 15237 15294 15309 1535315368 15413 15428 15485 15500 15557 15572 15615 15630 15687 15702 1581715832 15889 15904 15947 15962 548151 n/a n/a GGGCTTCAGCCAGACA eekd₁₀kke35 34238 34253 1691 548152 n/a n/a CGGGCTTCAGCCAGAC eekd₁₀kke 32 3423934254 1692 548153 2148 2163 TGCTGAAAGCGGGCTT eekd₁₀kke 44 34248 342631693 548154 2149 2164 GTGCTGAAAGCGGGCT eekd₁₀kke 7 34249 34264 1694548155 2150 2165 CGTGCTGAAAGCGGGC eekd₁₀kke 76 34250 34265 1695 5481562167 2182 TCAGCCCCTGGTTACG eekd₁₀kke 0 34267 34282 1696 548157 2171 2186ATTGTCAGCCCCTGGT eekd₁₀kke 7 34271 34286 1697 548158 2173 2188GCATTGTCAGCCCCTG eekd₁₀kke 18 34273 34288 1698 548159 2174 2189CGCATTGTCAGCCCCT eekd₁₀kke 59 34274 34289 1699 548160 2175 2190TCGCATTGTCAGCCCC eekd₁₀kke 60 34275 34290 1700 548161 2176 2191CTCGCATTGTCAGCCC eekd₁₀kke 59 34276 34291 1701 548162 2177 2192CCTCGCATTGTCAGCC eekd₁₀kke 25 34277 34292 1702 548163 2178 2193ACCTCGCATTGTCAGC eekd₁₀kke 46 34278 34293 1703 548164 2179 2194GACCTCGCATTGTCAG eekd₁₀kke 40 34279 34294 1704 548165 2180 2195CGACCTCGCATTGTCA eekd₁₀kke 53 34280 34295 1705 548166 2181 2196GCGACCTCGCATTGTC eekd₁₀kke 0 34281 34296 1706 548167 2182 2197TGCGACCTCGCATTGT eekd₁₀kke 36 34282 34297 1707 548168 2183 2198TTGCGACCTCGCATTG eekd₁₀kke 61 34283 34298 1708 548169 2184 2199GTTGCGACCTCGCATT eekd₁₀kke 7 34284 34299 1709 548170 2185 2200AGTTGCGACCTCGCAT eekd₁₀kke 68 34285 34300 1710 548171 2186 2201CAGTTGCGACCTCGCA eekd₁₀kke 47 34286 34301 1711 548172 2187 2202TCAGTTGCGACCTCGC eekd₁₀kke 0 34287 34302 1712 548173 2188 2203CTCAGTTGCGACCTCG eekd₁₀kke 51 34288 34303 1713 548174 2189 2204TCTCAGTTGCGACCTC eekd₁₀kke 68 34289 34304 1714 548175 2190 2205ATCTCAGTTGCGACCT eekd₁₀kke 0 34290 34305 1715 548176 2191 2206GATCTCAGTTGCGACC eekd₁₀kke 38 34291 34306 1716 548177 2192 2207AGATCTCAGTTGCGAC eekd₁₀kke 45 34292 34307 1717 548178 2193 2208GAGATCTCAGTTGCGA eekd₁₀kke 54 34293 34308 1718 548179 2194 2209GGAGATCTCAGTTGCG eekd₁₀kke 52 34294 34309 1719 548180 2198 2213TCATGGAGATCTCAGT eekd₁₀kke 79 34298 34313 1720 548181 2199 2214GTCATGGAGATCTCAG eekd₁₀kke 55 34299 34314 1721 548182 2200 2215AGTCATGGAGATCTCA eekd₁₀kke 55 34300 34315 1722 548183 2201 2216CAGTCATGGAGATCTC eekd₁₀kke 43 34301 34316 1723 548184 2202 2217ACAGTCATGGAGATCT eekd₁₀kke 73 34302 34317 1724 548185 2207 2222AACACACAGTCATGGA eekd₁₀kke 23 34307 34322 1725 548186 2208 2223CAACACACAGTCATGG eekd₁₀kke 0 34308 34323 1726 548187 n/a n/aCATCCTATCCGTGTTC eekd₁₀kke 33 3279 3294 1727 548189 n/a n/aCATGAACATCCTATCC eekd₁₀kke 24 3285 3300 1728 548190 n/a n/aTATTCCATGAACATCC eekd₁₀kke 43 3290 3305 1729 548191 n/a n/aGTCAACATATTCCATG eekd₁₀kke 0 3297 3312 1730 548192 n/a n/aCCTGTCAACATATTCC eekd₁₀kke 65 3300 3315 1731 548193 n/a n/aTGTCCTGTCAACATAT eekd₁₀kke 58 3303 3318 1732 548194 n/a n/aGCCAACAGTTTCAACT eekd₁₀kke 61 3322 3337 1733 548195 n/a n/aTTCTGCCAACAGTTTC eekd₁₀kke 84 3326 3341 1734 548196 n/a n/aCAATATTGACTTTGGG eekd₁₀kke 6 3343 3358 1735 548197 n/a n/aTGCTTGGCTTCAATAT eekd₁₀kke 68 3353 3368 1736 548198 n/a n/aACTGCAGGCAATATTT eekd₁₀kke 49 3369 3384 1737 548199 n/a n/aGCACTGCAGGCAATAT eekd₁₀kke 24 3371 3386 1738 548200 n/a n/aCTAATGTGGCACTGCA eekd₁₀kke 19 3379 3394 1739 548201 n/a n/aTGTTCTAATGTGGCAC eekd₁₀kke 67 3383 3398 1740 548202 n/a n/aGCTGTTCTAATGTGGC eekd₁₀kke 9 3385 3400 1741 548203 n/a n/aTGACTAGTGAATGGCT eekd₁₀kke 73 2280 2295 1742 548204 n/a n/aTCTGACTAGTGAATGG eekd₁₀kke 25 2282 2297 1743 548205 n/a n/aTCAATCTGACTAGTGA eekd₁₀kke 14 2286 2301 1744 548206 n/a n/aGGTCAATCTGACTAGT eekd₁₀kke 45 2288 2303 1745 548207 n/a n/aCTGGTCAATCTGACTA eekd₁₀kke 60 2290 2305 1746 548208 n/a n/aCTCTGGTCAATCTGAC eekd₁₀kke 19 2292 2307 1747 548209 n/a n/aCAATCTCTGGTCAATC eekd₁₀kke 57 2296 2311 1748 548210 n/a n/aCAACAATCTCTGGTCA eekd₁₀kke 55 2299 2314 1749 548211 n/a n/aACCAACAATCTCTGGT eekd₁₀kke 51 2301 2316 1750 548212 n/a n/aAGCCCACCAACAATCT eekd₁₀kke 44 2306 2321 1751 548213 n/a n/aGACAGCCCACCAACAA eekd₁₀kke 70 2309 2324 1752 548214 n/a n/aCAGACAGCCCACCAAC eekd₁₀kke 55 2311 2326 1753 548215 n/a n/aGCATAGACCCCAACAG eekd₁₀kke 61 2324 2339 1754 548216 n/a n/aGTGCATAGACCCCAAC eekd₁₀kke 45 2326 2341 1755 548217 n/a n/aCTGTGCATAGACCCCA eekd₁₀kke 69 2328 2343 1756 548218 n/a n/aTCCTGTGCATAGACCC eekd₁₀kke 59 2330 2345 1757 548219 n/a n/aGAAATCCTGTGCATAG eekd₁₀kke 8 2334 2349 1758 548220 n/a n/aGCAGAAATCCTGTGCA eekd₁₀kke 69 2337 2352 1759 548221 n/a n/aACTCCAGCAGAAATCC eekd₁₀kke 49 2343 2358 1760 548222 n/a n/aAATCATGCCTTGTGGG eekd₁₀kke 32 4765 4780 1761 548223 n/a n/aTAGACCCAGAATCATG eekd₁₀kke 50 4774 4789 1762 548224 n/a n/aCCATAGACCCAGAATC eekd₁₀kke 20 4777 4792 1763 548225 n/a n/aAGTCACCATAGACCCA eekd₁₀kke 48 4782 4797 1764 548226 n/a n/aTAAGTCACCATAGACC eekd₁₀kke 39 4784 4799 1765 548227 n/a n/aGTGGCCCTCTTAAGTC eekd₁₀kke 0 4794 4809 1766

TABLE 26 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 ISISStart Stop % Start Stop SEQ NO Site Site Sequence Chemistry inhibitionSite Site ID NO 531231 n/a n/a TATCACTGTACTAGTTTCCT eeeeed₁₀eeeee 4214744 14763 334 14815 14834 14886 14905 14945 14964 15005 15024 1507715096 15220 15239 15292 15311 15351 15370 15411 15430 15483 15502 1555515574 15613 15632 15685 15704 15815 15834 15887 15906 15945 15964 547747n/a n/a TCACTGTACTAGTTTC eekd₁₀kke 80 14746 14761 1267 14817 14832 1488814903 14947 14962 15007 15022 15079 15094 15222 15237 15294 15309 1535315368 15413 15428 15485 15500 15557 15572 15615 15630 15687 15702 1581715832 15889 15904 15947 15962 548228 n/a n/a GTTGTGTGGCCCTCTT eekd₁₀kke37 4799 4814 1767 548229 n/a n/a CATTGTTGTGTGGCCC eekd₁₀kke 31 4803 48181768 548230 n/a n/a TACTCATTGTTGTGTG eekd₁₀kke 10 4807 4822 1769 548231n/a n/a AATACTCATTGTTGTG eekd₁₀kke 11 4809 4824 1770 548232 n/a n/aGCCATACATCTGAGGA eekd₁₀kke 3 4831 4846 1771 548233 n/a n/aATTGTAGCCATACATC eekd₁₀kke 38 4837 4852 1772 548234 n/a n/aTTATTGTAGCCATACA eekd₁₀kke 17 4839 4854 1773 548235 n/a n/aTCTAGATGACCTGAAG eekd₁₀kke 0 18147 18162 1774 548236 n/a n/aTACATCTAGATGACCT eekd₁₀kke 37 18151 18166 1775 548237 n/a n/aGTATACATCTAGATGA eekd₁₀kke 22 18154 18169 1776 548238 n/a n/aACTCGCCTTTGTGACT eekd₁₀kke 31 26268 26283 1777 548239 n/a n/aTACTCGCCTTTGTGAC eekd₁₀kke 18 26269 26284 1778 548240 n/a n/aATACTCGCCTTTGTGA eekd₁₀kke 3 26270 26285 1779 26301 26316 548241 n/a n/aCATACTCGCCTTTGTG eekd₁₀kke 1 26271 26286 1780 26302 26317 548242 n/a n/aGCATACTCGCCTTTGT eekd₁₀kke 25 26272 26287 1781 26303 26318 548243 n/an/a ATGCATACTCGCCTTT eekd₁₀kke 0 26274 26289 1782 26305 26320 548244 n/an/a CATGCATACTCGCCTT eekd₁₀kke 51 26275 26290 1783 26306 26321 548245n/a n/a CCATGCATACTCGCCT eekd₁₀kke 31 26276 26291 1784 26307 26322548246 n/a n/a TTCCATGCATACTCGC eekd₁₀kke 46 26278 26293 1785 548247 n/an/a CGATTTTCCATGCATA eekd₁₀kke 56 26283 26298 1786 548248 n/a n/aTGCGATTTTCCATGCA eekd₁₀kke 13 26285 26300 1787 548249 n/a n/aTGTGATGCGATTTTCC eekd₁₀kke 22 26290 26305 1788 548250 n/a n/aCTTTGTGATGCGATTT eekd₁₀kke 0 26293 26308 1789 548251 n/a n/aGCCTTTGTGATGCGAT eekd₁₀kke 13 26295 26310 1790 548252 n/a n/aACTCGCCTTTGTGATG eekd₁₀kke 33 26299 26314 1791 548253 n/a n/aTACTCGCCTTTGTGAT eekd₁₀kke 8 26300 26315 1792 548254 n/a n/aCCCATGCATACTCGCC eekd₁₀kke 39 26308 26323 1793 548255 n/a n/aCCCCATGCATACTCGC eekd₁₀kke 38 26309 26324 1794 548256 n/a n/aGCTCCCCATGCATACT eekd₁₀kke 25 26312 26327 1795 548257 n/a n/aAGTGCTCCCCATGCAT eekd₁₀kke 2 26315 26330 1796 548258 n/a n/aCAAGTGCTCCCCATGC eekd₁₀kke 0 26317 26332 1797 548259 n/a n/aGTGATGAAAGTACAGC eekd₁₀kke 45 26335 26350 1798 548260 n/a n/aAGGAGTTTGTCAGAAC eekd₁₀kke 28 3210 3225 1799 548261 n/a n/aTTCAGGGAGTGATGTC eekd₁₀kke 36 3241 3256 1800 548262 n/a n/aCCTATCCGTGTTCAGC eekd₁₀kke 73 3276 3291 1801 548263 n/a n/aCTCTACATACTCAGGA eekd₁₀kke 62 3561 3576 1802 548264 n/a n/aCAGTCCAAAAATCCCT eekd₁₀kke 60 3701 3716 1803 548265 n/a n/aCCTCTTGATTTGGGCA eekd₁₀kke 85 3749 3764 1804 548266 n/a n/aTTGGCCAACTCTGTGG eekd₁₀kke 44 3816 3831 1805 548267 n/a n/aGACCTCCAGACTACTG eekd₁₀kke 34 3848 3863 1806 548268 n/a n/aTGTGTCTAGGGAGTTG eekd₁₀kke 52 3898 3913 1807 548269 n/a n/aAGCACACAATTACTGG eekd₁₀kke 62 3946 3961 1808 548270 n/a n/aCTGCTGGTTTTAGACC eekd₁₀kke 28 4029 4044 1809 548271 n/a n/aTTCACTTACCACAGGA eekd₁₀kke 56 4122 4137 1810 548272 n/a n/aGGTGCCACTTGCTTGG eekd₁₀kke 54 4178 4193 1811 548273 n/a n/aAATCTCCACCCCCGAA eekd₁₀kke 5 4224 4239 1812 548274 n/a n/aTACCTGACAAGTGGTC eekd₁₀kke 0 4287 4302 1813 548275 n/a n/aGTCCCAAGACATTCCT eekd₁₀kke 40 4350 4365 1814 548276 n/a n/aCAGAGTGTCATCTGCG eekd₁₀kke 49 4389 4404 1815 548277 n/a n/aGGATTGGACCCAGACA eekd₁₀kke 57 4511 4526 1816 548278 n/a n/aGGTTCCCTAGCGGTCC eekd₁₀kke 74 4564 4579 1817 548279 n/a n/aCACCTAGAACTATCCA eekd₁₀kke 39 4632 4647 1818 548280 n/a n/aCTCCCTCTGTAATGAT eekd₁₀kke 43 4736 4751 1819 548281 n/a n/aGGTTGAGGGACAGACA eekd₁₀kke 0 4944 4959 1820 548282 n/a n/aGTGGGTTTGCACATGG eekd₁₀kke 73 4992 5007 1821 548283 n/a n/aGGCTTATGCTCCTTCT eekd₁₀kke 56 5017 5032 1822 548284 n/a n/aCCCCCTGTAGTTGGCT eekd₁₀kke 35 5051 5066 1823 548285 n/a n/aGCTTACTTACATCCCT eekd₁₀kke 52 5132 5147 1824 548286 n/a n/aGGGACTACATGCAATA eekd₁₀kke 47 5166 5181 1825 548287 n/a n/aGTCAAAGAGTGTCCAC eekd₁₀kke 38 5283 5298 1826 548288 n/a n/aGAATAGCAAGCTCCAA eekd₁₀kke 64 5348 5363 1827 548289 n/a n/aCATGATACCACACCAC eekd₁₀kke 28 5484 5499 1828 548290 n/a n/aGAGCACTCTTATTAGC eekd₁₀kke 31 5546 5561 1829 548291 n/a n/aCCTGTTAGAGTTGGCC eekd₁₀kke 35 5576 5591 1830 548292 n/a n/aAGGACACTGTTTCCAG eekd₁₀kke 38 5627 5642 1831 548293 n/a n/aGTCACCAGAACCACAT eekd₁₀kke 44 5683 5698 1832 548294 n/a n/aGTGTGCACTTTCTGGT eekd₁₀kke 33 5716 5731 1833 548295 n/a n/aCTCTGATTGGGTCACC eekd₁₀kke 26 5746 5761 1834 548296 n/a n/aACCAACAACTCAGGCC eekd₁₀kke 34 5858 5873 1835 548297 n/a n/aACTCTCAAGCTCCACG eekd₁₀kke 32 5889 5904 1836 548298 n/a n/aGGACAATATGTCTCCT eekd₁₀kke 0 5935 5950 1837 548299 n/a n/aCATTGTGCTCAACTGA eekd₁₀kke 35 5961 5976 1838 548300 n/a n/aGCCCATGGTGAATCTG eekd₁₀kke 53 5995 6010 1839 548301 n/a n/aCCTAGTACAAAGTGGC eekd₁₀kke 65 6050 6065 1840 548302 n/a n/aGCCATTTTATCCCTGA eekd₁₀kke 71 6134 6149 1841 548303 n/a n/aGGGCCCCCATGTCCAT eekd₁₀kke 0 6336 6351 1842

TABLE 27 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 ISISStart Stop % Start Stop SEQ NO Site Site Sequence Chemistry inhibitionSite Site ID NO 531231 n/a n/a TATCACTGTACTAGTTTCCT eeeeed₁₀eeeee 7214744 14763 334 14815 14834 14886 14905 14945 14964 15005 15024 1507715096 15220 15239 15292 15311 15351 15370 15411 15430 15483 15502 1555515574 15613 15632 15685 15704 15815 15834 15887 15906 15945 15964 547747n/a n/a TCACTGTACTAGTTTC eekd₁₀kke 67 14746 14761 1267 14817 14832 1488814903 14947 14962 15007 15022 15079 15094 15222 15237 15294 15309 1535315368 15413 15428 15485 15500 15557 15572 15615 15630 15687 15702 1581715832 15889 15904 15947 15962 548305 n/a n/a GTTCTTGCTTATCCTC eekd₁₀kke55 6484 6499 1843 548306 n/a n/a ATGTGACAGTCAGGGA eekd₁₀kke 8 6559 65741844 548307 n/a n/a TTCTGCAACTGAGCCT eekd₁₀kke 6 6587 6602 1845 548308n/a n/a AATGGCAGGTCCTGGC eekd₁₀kke 9 6616 6631 1846 548309 n/a n/aAGACAGTTGGTGGTTT eekd₁₀kke 41 6700 6715 1847 548310 n/a n/aGAGGAGTTGGTTTAGT eekd₁₀kke 0 6750 6765 1848 548311 n/a n/aTGACCACCTCTCGGGT eekd₁₀kke 10 6860 6875 1849 548312 n/a n/aATTTGGCCCTGAGCCC eekd₁₀kke 0 6935 6950 1850 548313 n/a n/aGCCTTTGAGGGAGTGG eekd₁₀kke 35 7024 7039 1851 548314 n/a n/aACAACCTGTCCATTCC eekd₁₀kke 43 7087 7102 1852 548315 n/a n/aGTTGTCAACTGGGACC eekd₁₀kke 14 7125 7140 1853 548316 n/a n/aCTGTTCAGGTAGCACA eekd₁₀kke 64 7150 7165 1854 548317 n/a n/aCCGGGAAAGACTGTCT eekd₁₀kke 42 7190 7205 1855 548318 n/a n/aACTGCACCCCACATAT eekd₁₀kke 18 7257 7272 1856 548319 n/a n/aCCTCATCTCAGTATGA eekd₁₀kke 26 7398 7413 1857 548320 n/a n/aGCACACAGACTTGCCC eekd₁₀kke 0 7508 7523 1858 548321 n/a n/aCTGCATCTGGACTATG eekd₁₀kke 38 7559 7574 1859 548322 n/a n/aAGGGAAATTAGAGGCA eekd₁₀kke 38 7586 7601 1860 548323 n/a n/aCTGTTGCCTGACATGC eekd₁₀kke 43 7696 7711 1861 548324 n/a n/aACATAAATTCCCCACA eekd₁₀kke 29 7741 7756 1862 548325 n/a n/aCCCACTGACTGACTAC eekd₁₀kke 27 7906 7921 1863 548326 n/a n/aTCCTGTGACAGAACCA eekd₁₀kke 27 7988 8003 1864 548327 n/a n/aCTACACCTTTCTGCAC eekd₁₀kke 6 8221 8236 1865 548328 n/a n/aGGTCCTTGAACCCCGT eekd₁₀kke 68 8260 8275 1866 548329 n/a n/aAGCAGATCTGGGTTGT eekd₁₀kke 59 8328 8343 1867 548330 n/a n/aGACTAGCTTCTACTAC eekd₁₀kke 34 8404 8419 1868 548331 n/a n/aACAATCCCTTAGCCCA eekd₁₀kke 73 8457 8472 1869 548332 n/a n/aGATGAAATGTGCACCT eekd₁₀kke 46 8491 8506 1870 548333 n/a n/aGACTGTGCTATCCGCT eekd₁₀kke 58 8550 8565 1871 548334 n/a n/aGCTCACTATAGGCCCC eekd₁₀kke 69 8656 8671 1872 548335 n/a n/aTAGCATCATGCCACAG eekd₁₀kke 51 8684 8699 1873 548336 n/a n/aGCACATTAGGAGGTAG eekd₁₀kke 1 9039 9054 1874 548337 n/a n/aTACCGCTGGGTGCGGT eekd₁₀kke 10 9075 9090 1875 548338 n/a n/aATGAAACTGTGGCTCG eekd₁₀kke 80 9131 9146 1876 548339 n/a n/aACATGTGGGATCAGAG eekd₁₀kke 37 9275 9290 1877 548340 n/a n/aGATGATCCTCACATAC eekd₁₀kke 35 9316 9331 1878 548341 n/a n/aTAGAACCTTCCTCCAC eekd₁₀kke 30 9341 9356 1879 548342 n/a n/aGGAAGACTTCCCTCTG eekd₁₀kke 0 9403 9418 1880 548343 n/a n/aTAGTGATAAGAGCTGG eekd₁₀kke 78 9472 9487 1881 548344 n/a n/aGGCAACTATGTTCTCA eekd₁₀kke 76 9536 9551 1882 548345 n/a n/aCTAACTCCATCACTGC eekd₁₀kke 55 9637 9652 1883 548346 n/a n/aTCCCCAATACTTGCTG eekd₁₀kke 35 9696 9711 1884 548347 n/a n/aGCTGTTCTAAGCGAGA eekd₁₀kke 31 9976 9991 1885 548348 n/a n/aTGAGTGATGCCTTCCA eekd₁₀kke 82 10024 10039 1886 548349 n/a n/aTCCAGAATACTGCCCC eekd₁₀kke 61 10054 10069 1887 548350 n/a n/aGCGCTAACCTCATAAA eekd₁₀kke 29 10148 10163 1888 548351 n/a n/aCTGGAAACGAGACACA eekd₁₀kke 33 10201 10216 1889 548352 n/a n/aGAGAGAGATGTTCCCT eekd₁₀kke 47 10240 10255 1890 548353 n/a n/aCTGCTGGTTGAGAATC eekd₁₀kke 48 10287 10302 1891 548354 n/a n/aATGTCCCCAGTGGAAG eekd₁₀kke 41 10314 10329 1892 548355 n/a n/aGCATCCTCCCTAGTTG eekd₁₀kke 47 10362 10377 1893 548356 n/a n/aTGTTGGTCAGCATTCA eekd₁₀kke 63 10411 10426 1894 548357 n/a n/aGACGACTGCCCTGTGC eekd₁₀kke 69 10436 10451 1895 548358 n/a n/aATTTGGGCCTAGTGGT eekd₁₀kke 0 10515 10530 1896 548359 n/a n/aCCTAGTCCTCAAGTTT eekd₁₀kke 0 10580 10595 1897 548360 n/a n/aCAAGACATCAGTAGCT eekd₁₀kke 45 10626 10641 1898 548361 n/a n/aCTTATCAGTCCCAGTC eekd₁₀kke 52 10702 10717 1899 548362 n/a n/aGACAACCCATCAGTTG eekd₁₀kke 33 10742 10757 1900 548363 n/a n/aCAGCAGGCTCAAAGTG eekd₁₀kke 37 10915 10930 1901 548364 n/a n/aTGGCTAAGTCAGGCCC eekd₁₀kke 30 10982 10997 1902 548365 n/a n/aTGTACTCCACCTCACG eekd₁₀kke 55 11017 11032 1903 548366 n/a n/aAGCAAGCTAAGTGAGT eekd₁₀kke 5 11199 11214 1904 548367 n/a n/aGTTCTTGAGTGTAGAG eekd₁₀kke 52 11260 11275 1905 548368 n/a n/aGTGTTCATACGGAAGC eekd₁₀kke 59 11299 11314 1906 548369 n/a n/aGTTGGGATGCGACTCT eekd₁₀kke 50 11335 11350 1907 548370 n/a n/aACGAAGTCTCTTTCCT eekd₁₀kke 53 11385 11400 1908 548371 n/a n/aCGATGAGTTGGGCAGG eekd₁₀kke 57 11454 11469 1909 548372 n/a n/aGATACCTTTCCACTCC eekd₁₀kke 61 11558 11573 1910 548373 n/a n/aTCCCCAAGATTATGTG eekd₁₀kke 16 11596 11611 1911 548374 n/a n/aGCACCCTTTTCATTGA eekd₁₀kke 41 12074 12089 1912 548375 n/a n/aTCGACTTCTCCTGTCT eekd₁₀kke 27 12199 12214 1913 548376 n/a n/aGCCTTTGACCTTTCGC eekd₁₀kke 65 12261 12276 1914 548377 n/a n/aGTGTGCTGAGGTTTGC eekd₁₀kke 80 12297 12312 1915 548378 n/a n/aGCAAGATGCATGCAGC eekd₁₀kke 49 12393 12408 1916 548379 n/a n/aATCGAACTCTGCTTGA eekd₁₀kke 44 12477 12492 1917 548380 n/a n/aGCCCAGTTTTGGCAAC eekd₁₀kke 7 12540 12555 1918 548381 n/a n/aCCCACTACCATTTGGG eekd₁₀kke 0 12578 12593 1919

TABLE 28 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 ISISStart Stop % Start Stop SEQ NO Site Site Sequence Chemistry inhibitionSite Site ID NO 531231 n/a n/a TATCACTGTACTAGTTTCCT eeeeed₁₀eeeee 4614744 14763 334 14815 14834 14886 14905 14945 14964 15005 15024 1507715096 15220 15239 15292 15311 15351 15370 15411 15430 15483 15502 1555515574 15613 15632 15685 15704 15815 15834 15887 15906 15945 15964 547747n/a n/a TCACTGTACTAGTTTC eekd₁₀kke 64 14746 14761 1267 14817 14832 1488814903 14947 14962 15007 15022 15079 15094 15222 15237 15294 15309 1535315368 15413 15428 15485 15500 15557 15572 15615 15630 15687 15702 1581715832 15889 15904 15947 15962 548459 n/a n/a CAACTATAACAGTATC eekd₁₀kke26 15903 15918 1920 548460 n/a n/a CTATACCACGGTAACT eekd₁₀kke 0 1603616051 1921 548461 n/a n/a CCTATATCACTGTAAC eekd₁₀kke 0 16127 16142 1922548462 n/a n/a ACCTATATCACTGTAA eekd₁₀kke 0 16128 16143 1923 548463 n/an/a TCACTGTACCTATATC eekd₁₀kke 0 16135 16150 1924 548464 n/a n/aGTCCTATAACTATATC eekd₁₀kke 0 16174 16189 1925 548465 n/a n/aCTGTACCTATAACTGT eekd₁₀kke 0 16202 16217 1926 548466 n/a n/aCGTCACTGTACCTATA eekd₁₀kke 71 16207 16222 1927 548467 n/a n/aCATCACTGTACCTATA eekd₁₀kke 20 16258 16273 1928 548468 n/a n/aCAACATCACTGTACCT eekd₁₀kke 6 16261 16276 1929 548469 n/a n/aTTCCCTACCCCTGGTA eekd₁₀kke 0 16331 16346 1930 548470 n/a n/aGGTGGAATGTCATGGC eekd₁₀kke 56 16404 16419 1931 548471 n/a n/aGCGGAAAACTGGCCGT eekd₁₀kke 17 16474 16489 1932 548472 n/a n/aCCCAATACAGGGCCAG eekd₁₀kke 0 16513 16528 1933 548473 n/a n/aCCAACCTTCCCAATCT eekd₁₀kke 0 16554 16569 1934 548474 n/a n/aGAAGGTGTGCTGTCGC eekd₁₀kke 33 16602 16617 1935 548475 n/a n/aATCGAGTCCTGCCTCC eekd₁₀kke 17 16707 16722 1936 548476 n/a n/aGCAAATCCTTCCAGCA eekd₁₀kke 27 16755 16770 1937 548477 n/a n/aGCACGAGCTTGCCTGT eekd₁₀kke 26 16787 16802 1938 548478 n/a n/aGAGCCATCCAGGGTGC eekd₁₀kke 53 16845 16860 1939 548479 n/a n/aAGGCCATTTGATCCGA eekd₁₀kke 68 16913 16928 1940 548480 n/a n/aGCCACGCCCTTAGCAG eekd₁₀kke 20 16973 16988 1941 548481 n/a n/aGTTCCCTGAGGAACGG eekd₁₀kke 2 17010 17025 1942 548482 n/a n/aGGCAGTTAGGCCAGGA eekd₁₀kke 53 17068 17083 1943 548483 n/a n/aCTACAGATCATCCCTA eekd₁₀kke 5 17102 17117 1944 548484 n/a n/aCCCCGGAGCACCTTCA eekd₁₀kke 41 17207 17222 1945 548485 n/a n/aGTGACCCAAGGGTCGA eekd₁₀kke 17 17252 17267 1946 548486 n/a n/aCGTGGTTAGCCTGACA eekd₁₀kke 68 17416 17431 1947 548487 n/a n/aTCCATGTCAGAGTTGC eekd₁₀kke 71 17461 17476 1948 548488 n/a n/aCCTCCTTTTGGCTTGA eekd₁₀kke 63 17530 17545 1949 548489 n/a n/aTTCCCCAGAGGTGATA eekd₁₀kke 16 17582 17597 1950 548490 n/a n/aTCTGGTTAGCCTCCGA eekd₁₀kke 58 17664 17679 1951 548491 n/a n/aTGGCCAAGCAACCAGT eekd₁₀kke 57 17715 17730 1952 548492 n/a n/aGCCCAATGTCCTAACC eekd₁₀kke 51 17794 17809 1953 548493 n/a n/aCCACCGCTGCCCGCCA eekd₁₀kke 37 18013 18028 1954 548494 n/a n/aTGTGACCCCCCACCGC eekd₁₀kke 39 18022 18037 1955 548495 n/a n/aTTGTGACCCCCCACCG eekd₁₀kke 55 18023 18038 1956 548496 n/a n/aACTGAACCCCCTTAGG eekd₁₀kke 0 18571 18586 1957 548497 n/a n/aCCTTCATACCCCTCAC eekd₁₀kke 26 18725 18740 1958 548498 n/a n/aCCGATAACAGACCGGC eekd₁₀kke 71 18795 18810 1959 548499 n/a n/aATACCCGGAGTCAGGA eekd₁₀kke 56 18955 18970 1960 548500 n/a n/aATTGCTCAGGCCCCCT eekd₁₀kke 29 19037 19052 1961 548501 n/a n/aCAAGCCACTAACCCAC eekd₁₀kke 33 19147 19162 1962 548502 n/a n/aAATTCTTGGACCAAGG eekd₁₀kke 25 19234 19249 1963 548503 n/a n/aCCATCTACTCCCCCAT eekd₁₀kke 9 19291 19306 1964 548504 n/a n/aGCAGCGAGCATTCCAA eekd₁₀kke 28 19352 19367 1965 548505 n/a n/aGGACAATGCCTATGCT eekd₁₀kke 21 19386 19401 1966 548506 n/a n/aGAAGCCATTCACTGCA eekd₁₀kke 32 19436 19451 1967 548507 n/a n/aAAACTCCTCTCAAGGC eekd₁₀kke 53 19474 19489 1968 548508 n/a n/aGCACCACCATGCGGTT eekd₁₀kke 43 19553 19568 1969 548509 n/a n/aTGCAGGGCTGCGCAGT eekd₁₀kke 41 19960 19975 1970 548510 n/a n/aTTAGCCACTCCTCTTG eekd₁₀kke 30 20062 20077 1971 548511 n/a n/aAGCTAGCTGACCCCAA eekd₁₀kke 16 20092 20107 1972 548512 n/a n/aTCCGCCTTTGGATACT eekd₁₀kke 49 20155 20170 1973 548513 n/a n/aCCTGCTGATTGTGTCT eekd₁₀kke 16 20240 20255 1974 548514 n/a n/aTCGAGGACAGCCCCCA eekd₁₀kke 40 20335 20350 1975 548515 n/a n/aACCCGTCAGCCTCAGC eekd₁₀kke 59 20381 20396 1976 548516 n/a n/aCTTGCCTATTCACCCC eekd₁₀kke 49 20544 20559 1977 548517 n/a n/aCGGACAAGCCTTACAG eekd₁₀kke 43 20596 20611 1978 548518 n/a n/aCACACTTACCCCGCTC eekd₁₀kke 12 20741 20756 1979 548519 n/a n/aCCTCCCCTTGTGTGTC eekd₁₀kke 31 20843 20858 1980 548520 n/a n/aCCGCTTCCCTGACTGT eekd₁₀kke 43 20919 20934 1981 548521 n/a n/aCAGCTCCCTTACTAGG eekd₁₀kke 61 20958 20973 1982 548522 n/a n/aAGGTATTGACCGCCAG eekd₁₀kke 55 21062 21077 1983 548523 n/a n/aGGTAAATCCATCCCCT eekd₁₀kke 44 21157 21172 1984 548524 n/a n/aGCCCGATCACCTTAGA eekd₁₀kke 45 21220 21235 1985 548525 n/a n/aGTCTAACTGGCCTGGC eekd₁₀kke 2 21328 21343 1986 548526 n/a n/aCTAAGCTGTGTCTCAT eekd₁₀kke 26 21373 21388 1987 548527 n/a n/aTGTTTCAAGTGCCAGA eekd₁₀kke 50 21434 21449 1988 548528 n/a n/aTGCAGTGGTCAAGCAT eekd₁₀kke 32 21478 21493 1989 548529 n/a n/aGCGATTCCTTGCCTCT eekd₁₀kke 56 21554 21569 1990 548530 n/a n/aATAATAGAGGCAGCCA eekd₁₀kke 50 21592 21607 1991 548531 n/a n/aGTCAGAAGGCCTCTTA eekd₁₀kke 21 21753 21768 1992 548532 n/a n/aTATTTATCCGACCTCT eekd₁₀kke 34 21881 21896 1993 548533 n/a n/aGAGGTGGTTGGAGCTA eekd₁₀kke 9 21926 21941 1994 548534 n/a n/aCAGATCCCAATTCTTC eekd₁₀kke 22 22063 22078 1995 548535 n/a n/aGAGTCTTTCCAATCCT eekd₁₀kke 13 22142 22157 1996

TABLE 29 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 SEQ ISISStart Stop % Start Stop ID NO Site Site Sequence Chemistry inhibitionSite Site NO 531231 n/a n/a TATCACTGTACTAGTTTCCT eeeeed₁₀eeeee 46 1474414763 334 14815 14834 14886 14905 14945 14964 15005 15024 15077 1509615220 15239 15292 15311 15351 15370 15411 15430 15483 15502 15555 1557415613 15632 15685 15704 15815 15834 15887 15906 15945 15964 547747 n/an/a TCACTGTACTAGTTTC eekd₁₀kke 64 14746 14761 1267 14817 14832 1488814903 14947 14962 15007 15022 15079 15094 15222 15237 15294 15309 1535315368 15413 15428 15485 15500 15557 15572 15615 15630 15687 15702 1581715832 15889 15904 15947 15962 548536 n/a n/a TCTCAATCCCAACCCC eekd₁₀kke0 22168 22183 1997 548537 n/a n/a CCTCAATCCCAACCCA eekd₁₀kke 0 2219122206 1998 548538 n/a n/a TAGTGGCAAGAACCAC eekd₁₀kke 0 22627 22642 1999548539 n/a n/a CGCGCGAATGCCTGCC eekd₁₀kke 41 22658 22673 2000 548540 n/an/a GACACCTGCTTGATTA eekd₁₀kke 7 22704 22719 2001 548541 n/a n/aGGCACTGGTCATGGAC eekd₁₀kke 39 22760 22775 2002 548542 n/a n/aGCGCCATCCTTCAATC eekd₁₀kke 7 22857 22872 2003 548543 n/a n/aGATCCACCCATGACCT eekd₁₀kke 32 22997 23012 2004 548544 n/a n/aGCTGTGACTCAGATCA eekd₁₀kke 62 23070 23085 2005 548545 n/a n/aCTCTTCGCATGGACAC eekd₁₀kke 46 23100 23115 2006 548546 n/a n/aGCCCAAGCCTACATGC eekd₁₀kke 35 23430 23445 2007 548547 n/a n/aGTGCGATTAAGCCCCA eekd₁₀kke 86 23514 23529 2008 548548 n/a n/aGCTTGTAGAAGGGATT eekd₁₀kke 54 23631 23646 2009 548549 n/a n/aTGTGCAATCAGGTGGA eekd₁₀kke 56 23765 23780 2010 548550 n/a n/aCCGGCCTGGATACAGC eekd₁₀kke 0 23831 23846 2011 548551 n/a n/aCGGCCAATGGGAAAGG eekd₁₀kke 25 24175 24190 2012 548552 n/a n/aTGGAGGAGTAGGGAAT eekd₁₀kke 10 24200 24215 2013 548553 n/a n/aCCCGAAGAGTCAAGTC eekd₁₀kke 46 24255 24270 2014 548554 n/a n/aGTGCTGCATTGCATGA eekd₁₀kke 42 24290 24305 2015 548555 n/a n/aACACGCCAGGTGAAAA eekd₁₀kke 2 24322 24337 2016 548556 n/a n/aATGCATGCCTACCCAA eekd₁₀kke 43 24526 24541 2017 548557 n/a n/aGTTACTCTGTGATCCA eekd₁₀kke 81 24581 24596 2018 548558 n/a n/aAACATTGTGTAGCTGC eekd₁₀kke 75 24640 24655 2019 548559 n/a n/aGAGACTGAAGCCCTCA eekd₁₀kke 44 24676 24691 2020 548560 n/a n/aCACTGCCTAGAAAGGC eekd₁₀kke 16 24734 24749 2021 548561 n/a n/aTGTAGTATCCAGAGTA eekd₁₀kke 46 24930 24945 2022 548562 n/a n/aAGATGACCTGCAGATG eekd₁₀kke 50 24983 24998 2023 548563 n/a n/aAAACCATGAATTAGGT eekd₁₀kke 20 25100 25115 2024 548564 n/a n/aTTGCTACTTTACACCA eekd₁₀kke 69 25208 25223 2025 548565 n/a n/aGGCATTAGGATAGGCA eekd₁₀kke 63 25350 25365 2026 548566 n/a n/aCACTCAGACTGTCTGA eekd₁₀kke 0 25413 25428 2027 548567 n/a n/aAGATCCGGAATAACCA eekd₁₀kke 67 25459 25474 2028 548568 n/a n/aATTGACAACCATCCTA eekd₁₀kke 27 25496 25511 2029 548569 n/a n/aACTCATTGGTCTACAG eekd₁₀kke 41 25559 25574 2030 548570 n/a n/aATGCCTTGTGCCTATT eekd₁₀kke 74 25706 25721 2031 548571 n/a n/aACTCTGAGGCCTTAGG eekd₁₀kke 59 25794 25809 2032 548572 n/a n/aGCATTACTCAGCATGT eekd₁₀kke 63 25836 25851 2033 548573 n/a n/aCCAGTCACCACCATTG eekd₁₀kke 65 25862 25877 2034 548574 n/a n/aGGTCTAACTCTAAGGG eekd₁₀kke 0 25920 25935 2035 548575 n/a n/aTGTCCTTTAAAGTATC eekd₁₀kke 18 25971 25986 2036 548576 n/a n/aTCATGTGGCAACCTGT eekd₁₀kke 41 26114 26129 2037 548577 n/a n/aAATCTGCACCTGGCAG eekd₁₀kke 42 26428 26443 2038 548578 n/a n/aCATGGCTATTGCTTCC eekd₁₀kke 73 26513 26528 2039 548579 n/a n/aGGGCTATATTGCCAGC eekd₁₀kke 46 26614 26629 2040 548580 n/a n/aCCAGAGCCTTGATCAG eekd₁₀kke 36 26681 26696 2041 548581 n/a n/aGGTGGGTTATCTGAGA eekd₁₀kke 13 26710 26725 2042 548582 n/a n/aTAGCTCCATGCTGTGT eekd₁₀kke 59 26735 26750 2043 548583 n/a n/aGGGAATTTATGCTGCC eekd₁₀kke 79 26782 26797 2044 548584 n/a n/aTGATGAAGTTCCACCT eekd₁₀kke 47 26840 26855 2045 548585 n/a n/aTAGGCACAGACAACCT eekd₁₀kke 33 26869 26884 2046 548586 n/a n/aTCCAACTACAGGACTC eekd₁₀kke 39 26943 26958 2047 548587 n/a n/aTTCTGGGAAACTCTCT eekd₁₀kke 45 26969 26984 2048 548588 n/a n/aAGCTCACACCCAAAAA eekd₁₀kke 10 27006 27021 2049 548589 n/a n/aTCTGTTACCTTGAGGA eekd₁₀kke 40 27280 27295 2050 548590 n/a n/aTGGTCATGTCAACTGT eekd₁₀kke 35 27550 27565 2051 548591 n/a n/aGTAAGCCTTCACAGGG eekd₁₀kke 3 27583 27598 2052 548592 n/a n/aCTCACCAGAGTTGTCC eekd₁₀kke 7 27726 27741 2053 548593 n/a n/aCATCCCTGACAGGTCC eekd₁₀kke 61 27759 27774 2054 548594 n/a n/aCCCTTCTAACCAAGGA eekd₁₀kke 30 27825 27840 2055 548595 n/a n/aGGATGAGATGCATCCA eekd₁₀kke 8 28069 28084 2056 548596 n/a n/aATGGCGGTGAAGCAGC eekd₁₀kke 20 28127 28142 2057 548597 n/a n/aTGAATACCATCCCCGC eekd₁₀kke 50 28171 28186 2058 548598 n/a n/aGCGCCATCTGCCCTGT eekd₁₀kke 50 28253 28268 2059 548599 n/a n/aTGGGTTGGAGGAGTGG eekd₁₀kke 19 28311 28326 2060 548600 n/a n/aTGGTGGTGGGATTGGT eekd₁₀kke 53 28336 28351 2061 28391 28406 28434 2844928446 28461 28525 28540 28611 28626 28623 28638 548601 n/a n/aTTGGTGGTGGGATTGG eekd₁₀kke 18 28337 28352 2062 28392 28407 28435 2845028447 28462 28526 28541 28612 28627 28624 28639 548602 n/a n/aGGTGGTGGAATTGGTG eekd₁₀kke 20 28347 28362 2063 548603 n/a n/aGAGATTGGTGGTGGGT eekd₁₀kke 35 28372 28387 2064 548604 n/a n/aGTGGTGGGATTGGTGC eekd₁₀kke 22 28432 28447 2065 548605 n/a n/aTGGCGGGATTGGTGGT eekd₁₀kke 12 28479 28494 2066 28558 28573 548606 n/an/a CGGTGGTGGGATTGGT eekd₁₀kke 41 28501 28516 2067 28580 28595 548607n/a n/a TCGGTGGTGGGATTGG eekd₁₀kke 34 28502 28517 2068 28581 28596548608 n/a n/a ATCGGTGGTGGGATTG eekd₁₀kke 25 28503 28518 2069 2858228597 548609 n/a n/a GATCGGTGGTGGGATT eekd₁₀kke 30 28504 28519 207028583 28598 548610 n/a n/a GGATCGGTGGTGGGAT eekd₁₀kke 2 28505 28520 207128584 28599 548611 n/a n/a GCGGGATCGGTGGTGG eekd₁₀kke 7 28508 28523 207228587 28602 548612 n/a n/a GGCGGGATCGGTGGTG eekd₁₀kke 20 28509 285242073 28588 28603

TABLE 30 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 ISISStart Stop % Start Stop SEQ NO Site Site Sequence Chemistry inhibitionSite Site ID NO 531231 n/a n/a TATCACTGTACTAGTTTCCT eeeeed₁₀eeeee 4614744 14763 334 14815 14834 14886 14905 14945 14964 15005 15024 1507715096 15220 15239 15292 15311 15351 15370 15411 15430 15483 15502 1555515574 15613 15632 15685 15704 15815 15834 15887 15906 15945 15964 547747n/a n/a TCACTGTACTAGTTTC eekd₁₀kke 64 14746 14761 1267 14817 14832 1488814903 14947 14962 15007 15022 15079 15094 15222 15237 15294 15309 1535315368 15413 15428 15485 15500 15557 15572 15615 15630 15687 15702 1581715832 15889 15904 15947 15962 548382 n/a n/a GAGCAAATACAGTCCA eekd₁₀kke19 12620 12635 2074 548383 n/a n/a GTCTCGATGGCAAGCT eekd₁₀kke 49 1265412669 2075 548384 n/a n/a CTCACCGGTACTCTGC eekd₁₀kke 49 12805 12820 2076548385 n/a n/a TCCTGGAGGCACCAAT eekd₁₀kke 0 12847 12862 2077 548386 n/an/a AGCCCTGTTTGGTTTT eekd₁₀kke 0 12903 12918 2078 548387 n/a n/aTGAAGGGCGAGGCGCA eekd₁₀kke 22 13261 13276 2079 548388 n/a n/aAAGAGGATGTCAGGCT eekd₁₀kke 4 13357 13372 2080 548389 n/a n/aTTGAGGAAAGACCTGC eekd₁₀kke 11 13399 13414 2081 548390 n/a n/aGCTGAGTGTGACTTAA eekd₁₀kke 43 13455 13470 2082 548391 n/a n/aGTACATGACTCCAGTG eekd₁₀kke 34 13638 13653 2083 548392 n/a n/aGTAGAGCATGGAGCGA eekd₁₀kke 31 13730 13745 2084 548393 n/a n/aCGCTTCAGGAAAGCGA eekd₁₀kke 26 13828 13843 2085 548394 n/a n/aGGCAGGAGACTCCGTG eekd₁₀kke 25 13919 13934 2086 548395 n/a n/aATCCTTCCCCTCGCAA eekd₁₀kke 0 13966 13981 2087 548396 n/a n/aTAATGAGTGGGTTAGG eekd₁₀kke 0 14007 14022 2088 548397 n/a n/aGGAGCAGTGCAGGTAA eekd₁₀kke 1 14065 14080 2089 548398 n/a n/aATAGGCAATTGTTCCT eekd₁₀kke 55 14129 14144 2090 548399 n/a n/aAGTCCTACAATTACCA eekd₁₀kke 11 14239 14254 2091 548400 n/a n/aGGGCTCCTATTCCACC eekd₁₀kke 13 14277 14292 2092 548401 n/a n/aGCCAGCTATGGGAACA eekd₁₀kke 71 14333 14348 2093 548402 n/a n/aCCCCATCTCGAAGCCC eekd₁₀kke 45 14380 14395 2094 548403 n/a n/aGAGTACATTGGGCCCA eekd₁₀kke 25 14418 14433 2095 548404 n/a n/aGAGCCTTCCGCCTCTC eekd₁₀kke 37 14471 14486 2096 548405 n/a n/aCGGACCTTCATCTTCA eekd₁₀kke 35 14529 14544 2097 548406 n/a n/aTCTAGAGGCCGCCTGC eekd₁₀kke 0 14558 14573 2098 548407 n/a n/aCCTATAACTGCTGCTC eekd₁₀kke 24 14731 14746 2099 548408 n/a n/aTATCACTGTACTAGTT eekd₁₀kke 47 14748 14763 1269 14819 14834 14890 1490514949 14964 15009 15024 15081 15096 15153 15168 15224 15239 15296 1531115355 15370 15415 15430 15487 15502 15559 15574 15617 15632 15689 1570415819 15834 15891 15906 15949 15964 548409 n/a n/a GTATCACTGTACTAGTeekd₁₀kke 81 14749 14764 2100 14820 14835 14891 14906 14950 14965 1501015025 15082 15097 15154 15169 15225 15240 15297 15312 15356 15371 1541615431 15488 15503 15560 15575 15618 15633 15690 15705 15820 15835 1589215907 15950 15965 548410 n/a n/a AGTATCACTGTACTAG eekd₁₀kke 85 1475014765 2101 14821 14836 14892 14907 14951 14966 15011 15026 15083 1509815155 15170 15226 15241 15298 15313 15357 15372 15417 15432 15489 1550415561 15576 15619 15634 15691 15706 15821 15836 15893 15908 15951 15966548411 n/a n/a CAGTATCACTGTACTA eekd₁₀kke 72 14751 14766 2102 1482214837 14893 14908 14952 14967 15012 15027 15084 15099 15156 15171 1522715242 15299 15314 15358 15373 15418 15433 15490 15505 15562 15577 1562015635 15692 15707 15822 15837 15894 15909 15952 15967 548412 n/a n/aTAACAGTATCACTGTA eekd₁₀kke 17 14754 14769 2103 14825 14840 14896 1491114955 14970 15015 15030 15087 15102 15159 15174 15230 15245 15302 1531715361 15376 15421 15436 15493 15508 15565 15580 15623 15638 15695 1571015825 15840 15897 15912 15955 15970 548413 n/a n/a CTAACAGTATCACTGTeekd₁₀kke 55 14755 14770 2104 14826 14841 14897 14912 15016 15031 1508815103 15231 15246 15303 15318 15422 15437 15494 15509 15624 15639 1582615841 15956 15971 548414 n/a n/a TCTAACAGTATCACTG eekd₁₀kke 20 1475614771 2105 14827 14842 14898 14913 15017 15032 15089 15104 15232 1524715304 15319 15423 15438 15495 15510 15625 15640 15827 15842 15957 15972548415 n/a n/a ATAACTCTAACAGTAT eekd₁₀kke 0 14761 14776 2106 14832 1484714903 14918 15022 15037 15094 15109 15237 15252 15309 15324 15428 1544315500 15515 15630 15645 15832 15847 15962 15977 548416 n/a n/aCTATAACTCTAACAGT eekd₁₀kke 9 14763 14778 2107 14834 14849 14905 1492015024 15039 15096 15111 15239 15254 15311 15326 15430 15445 15502 1551715632 15647 15834 15849 15964 15979 548417 n/a n/a ACTGTCCTATAACTCTeekd₁₀kke 24 14769 14784 2108 14840 14855 548418 n/a n/aTATATCACTGTCCTAT eekd₁₀kke 39 14775 14790 2109 14846 14861 15180 1519515716 15731 16164 16179 548419 n/a n/a CCTATATCACTGTCCT eekd₁₀kke 5214777 14792 2110 14848 14863 15182 15197 15718 15733 548420 n/a n/aTCCTATATCACTGTCC eekd₁₀kke 58 14778 14793 2111 14849 14864 15183 1519815719 15734 548421 n/a n/a CACTGTCCTATATCAC eekd₁₀kke 56 14783 147982112 14854 14869 14979 14994 15117 15132 15188 15203 15260 15275 1538515400 15523 15538 15653 15668 15724 15739 15855 15870 15985 16000 548422n/a n/a GTATCACTGTCCTATA eekd₁₀kke 69 14787 14802 2113 14983 14998 1512115136 15389 15404 15527 15542 15989 16004 548423 n/a n/aAGTATCACTGTCCTAT eekd₁₀kke 72 14788 14803 2114 14984 14999 15050 1506515122 15137 15390 15405 15456 15471 15528 15543 15990 16005 548424 n/an/a CAGTATCACTGTCCTA eekd₁₀kke 90 14789 14804 2115 14985 15000 1505115066 15123 15138 15391 15406 15457 15472 15529 15544 15991 16006 548425n/a n/a AACAGTATCACTGTCC eekd₁₀kke 90 14791 14806 2116 14987 15002 1505315068 15125 15140 15393 15408 15459 15474 15531 15546 15993 16008 548426n/a n/a TATAACAGTATCACTG eekd₁₀kke 14 14794 14809 2117 14990 15005 1505615071 15128 15143 15161 15176 15363 15378 15396 15411 15462 15477 1553415549 15567 15582 15697 15712 15899 15914 15996 16011 548427 n/a n/aCTATAACAGTATCACT eekd₁₀kke 24 14795 14810 2118 14991 15006 15057 1507215129 15144 15162 15177 15364 15379 15397 15412 15463 15478 15535 1555015568 15583 15698 15713 15900 15915 15997 16012 548428 n/a n/aTAACTATAACAGTATC eekd₁₀kke 0 14798 14813 2119 15060 15075 15132 1514715165 15180 15466 15481 15538 15553 15571 15586 15701 15716 15772 1578716000 16015 548429 n/a n/a TATAACTATAACAGTA eekd₁₀kke 0 14800 14815 212015062 15077 15134 15149 15167 15182 15468 15483 15540 15555 15573 1558815703 15718 15774 15789 16002 16017 548430 n/a n/a CCTATAACTATAACAGeekd₁₀kke 21 14802 14817 2121 15064 15079 15169 15184 15470 15485 1554215557 15575 15590 15705 15720 15776 15791 16004 16019 548431 n/a n/aTACCTATAACTCTAAC eekd₁₀kke 9 14908 14923 2122 15027 15042 15099 1511415242 15257 15314 15329 15433 15448 15505 15520 15635 15650 15837 1585215967 15982 548432 n/a n/a ACTGTACCTATAACTC eekd₁₀kke 43 14912 149272123 15031 15046 15246 15261 15318 15333 15437 15452 15509 15524 1563915654 15841 15856 15971 15986 548433 n/a n/a TATCACTGTACCTATA eekd₁₀kke33 14916 14931 2124 15250 15265 15322 15337 15375 15390 15513 1552815643 15658 15786 15801 15845 15860 15975 15990 16137 16152 548434 n/an/a ACAATATCACTGTACC eekd₁₀kke 63 14920 14935 2125 15326 15341 1579015805 16063 16078 16141 16156 548435 n/a n/a AACAATATCACTGTAC eekd₁₀kke19 14921 14936 2126 15327 15342 15791 15806 16064 16079 16142 16157548436 n/a n/a ATATCACTGTACCTGT eekd₁₀kke 8 14970 14985 2127 548437 n/an/a TATATCACTGTACCTG eekd₁₀kke 74 14971 14986 2128 548438 n/a n/aCTATATCACTGTACCT eekd₁₀kke 38 14972 14987 2129 15253 15268 15378 1539315516 15531 15646 15661 15848 15863 15978 15993 548439 n/a n/aCCTATATCACTGTACC eekd₁₀kke 46 14973 14988 2130 15254 15269 15379 1539415517 15532 15647 15662 15849 15864 15979 15994 548440 n/a n/aCCTATAACAGTATCAC eekd₁₀kke 32 14992 15007 2131 15365 15380 15398 15413548441 n/a n/a TCCTATAACAGTATCA eekd₁₀kke 42 14993 15008 2132 1539915414 548442 n/a n/a TTCCTATAACAGTATC eekd₁₀kke 17 14994 15009 213315400 15415 548443 n/a n/a GTTTCCTATAACAGTA eekd₁₀kke 12 14996 150112134 15402 15417 548444 n/a n/a CTATGTCACTGTACCT eekd₁₀kke 43 1503815053 2135 15444 15459 548445 n/a n/a CCTATGTCACTGTACC eekd₁₀kke 6215039 15054 2136 15445 15460 548446 n/a n/a TCCTATGTCACTGTAC eekd₁₀kke16 15040 15055 2137 15446 15461 548447 n/a n/a CACTGTCCTATGTCACeekd₁₀kke 59 15045 15060 2138 15451 15466 548448 n/a n/aTCACTGTCCTATGTCA eekd₁₀kke 61 15046 15061 2139 15452 15467 548449 n/an/a ATCACTGTCCTATGTC eekd₁₀kke 62 15047 15062 2140 15453 15468 548450n/a n/a CTACCTATAACTCTAA eekd₁₀kke 0 15100 15115 2141 548451 n/a n/aGTCCTATAACTATAAC eekd₁₀kke 0 15171 15186 2142 15577 15592 15707 1572216006 16021 16077 16092 16102 16117 16155 16170 548452 n/a n/aTATATCACTGTACCTA eekd₁₀kke 65 15252 15267 2143 15377 15392 15515 1553015645 15660 15847 15862 15977 15992 548453 n/a n/a TACCTATAACAGTATCeekd₁₀kke 12 15367 15382 2144 548454 n/a n/a ACTGTACCTATAACAG eekd₁₀kke17 15371 15386 2145 548455 n/a n/a CACCGTACTAGTTTCC eekd₁₀kke 64 1575715772 2146 548456 n/a n/a TATAACAGTATCACCG eekd₁₀kke 52 15768 15783 2147548457 n/a n/a CTATAACAGTATCACC eekd₁₀kke 13 15769 15784 2148 548458 n/an/a ACCTATAACTATAACA eekd₁₀kke 0 15777 15792 2149 16249 16264

TABLE 31 SEQ SEQ SEQ SEQ ID ID ID ID NO: NO: NO: 1 NO: 1 10 10 SEQ ISISStart Stop % Start Stop ID NO Site Site Sequence Chemistry inhibitionSite Site NO 531231 n/a n/a TATCACTGTACTAGTTTCCT eeeeed₁₀eeeee 48 1474414763 334 14815 14834 14886 14905 14945 14964 15005 15024 15077 1509615220 15239 15292 15311 15351 15370 15411 15430 15483 15502 15555 1557415613 15632 15685 15704 15815 15834 15887 15906 15945 15964 547747 n/an/a TCACTGTACTAGTTTC eekd₁₀kke 88 14746 14761 1267 14817 14832 1488814903 14947 14962 15007 15022 15079 15094 15222 15237 15294 15309 1535315368 15413 15428 15485 15500 15557 15572 15615 15630 15687 15702 1581715832 15889 15904 15947 15962 548613 n/a n/a TGGCGGGATCGGTGGT eekd₁₀kke39 28510 28525 2150 28589 28604 548614 n/a n/a TGGTGGCGGGATCGGTeekd₁₀kke 0 28513 28528 2151 28592 28607 548615 n/a n/a TTGGTGGCGGGATCGGeekd₁₀kke 10 28514 28529 2152 28593 28608 548616 n/a n/aATTGGTGGCGGGATCG eekd₁₀kke 35 28515 28530 2153 548617 n/a n/aGATTGGTGGCGGGATC eekd₁₀kke 44 28516 28531 2154 548618 n/a n/aGTTGGTGGCGGGATCG eekd₁₀kke 18 28594 28609 2155 548619 n/a n/aGGTTGGTGGCGGGATC eekd₁₀kke 19 28595 28610 2156 548620 n/a n/aTGGTTGGTGGCGGGAT eekd₁₀kke 24 28596 28611 2157 548621 n/a n/aGAACACATCAGGGATT eekd₁₀kke 33 28638 28653 2158 548622 n/a n/aTTTCTATGGGCCTGAC eekd₁₀kke 0 28669 28684 2159 548623 n/a n/aGCTGTCACTTAAGCCA eekd₁₀kke 16 28862 28877 2160 548624 n/a n/aTCTAGGGCCACACCTC eekd₁₀kke 24 28892 28907 2161 548625 n/a n/aGTTCTACACACAGTAC eekd₁₀kke 0 29014 29029 2162 548626 n/a n/aGCAGTATGTTCAATCC eekd₁₀kke 36 29202 29217 2163 548627 n/a n/aCCCACATGTACCACCG eekd₁₀kke 22 29235 29250 2164 548628 n/a n/aGTATGGCAGAGCCCCT eekd₁₀kke 9 29285 29300 2165 548629 n/a n/aCCCATCTTGGGACTTT eekd₁₀kke 44 29341 29356 2166 548630 n/a n/aTGGTCCCAAATTGGAG eekd₁₀kke 33 29387 29402 2167 548631 n/a n/aCTCACAATACTGAGCC eekd₁₀kke 55 29421 29436 2168 548632 n/a n/aGGAGATATCAGGTGCA eekd₁₀kke 45 29499 29514 2169 548633 n/a n/aCAAGGCATGTGTGCAC eekd₁₀kke 41 29534 29549 2170 548634 n/a n/aGCCTTATTCTGTGCAA eekd₁₀kke 0 29583 29598 2171 548635 n/a n/aAGGTGTGGCGCGCGCC eekd₁₀kke 18 29853 29868 2172 548636 n/a n/aCTCTATACAGCTGGGC eekd₁₀kke 5 30000 30015 2173 548637 n/a n/aGCTGATCTTCTAATGC eekd₁₀kke 38 30063 30078 2174 548638 n/a n/aCCTCATTGCTCCACTA eekd₁₀kke 26 30103 30118 2175 548639 n/a n/aTGGGAAGAAACTAGCA eekd₁₀kke 10 30159 30174 2176 548640 n/a n/aGAATGTTGCTGTCCCA eekd₁₀kke 32 30194 30209 2177 548641 n/a n/aGCATCATGCTTACTGC eekd₁₀kke 23 30225 30240 2178 548642 n/a n/aGCGGCAGTAGTGAATC eekd₁₀kke 23 30288 30303 2179 548643 n/a n/aCCTACCTAATTCCTCC eekd₁₀kke 0 30329 30344 2180 548644 n/a n/aAACTGGGCAGTCCTTC eekd₁₀kke 14 30418 30433 2181 548645 n/a n/aCCAGCGCAATTCTGCT eekd₁₀kke 8 30666 30681 2182 548646 n/a n/aCGTTTCCCTCAACTCC eekd₁₀kke 24 30750 30765 2183 548647 n/a n/aCACGGCAAGTCGCGGG eekd₁₀kke 39 30790 30805 2184 548648 n/a n/aCAGTTGTATCCCTCCC eekd₁₀kke 32 30852 30867 2185 548649 n/a n/aGCCTCTCAGACGGCAC eekd₁₀kke 0 30906 30921 2186 548650 n/a n/aCTGATCCCACTTGCCC eekd₁₀kke 21 30991 31006 2187 548651 n/a n/aAGTCTCTTTCCTACCC eekd₁₀kke 61 31030 31045 2188 548652 n/a n/aCCACGATGCTCTGGCC eekd₁₀kke 65 31068 31083 2189 548653 n/a n/aTCGGCTCCTGGCAGCA eekd₁₀kke 46 31111 31126 2190 548654 n/a n/aACCATTCCTGACCATG eekd₁₀kke 34 31151 31166 2191 548655 n/a n/aCCCGAGGTCACATAAT eekd₁₀kke 56 31416 31431 2192 548656 n/a n/aTTACAACAGACCCAGG eekd₁₀kke 35 31497 31512 2193 548657 n/a n/aAGCAGGGTATCTTCAC eekd₁₀kke 26 31548 31563 2194 548658 n/a n/aGAAGTTCCTGTGTCTT eekd₁₀kke 11 31593 31608 2195 548659 n/a n/aCCAACCTCTAAGGCTA eekd₁₀kke 17 31721 31736 2196 548660 n/a n/aATGCTTACCTTTCTCC eekd₁₀kke 0 31955 31970 2197 548661 n/a n/aACGACCCACTCCATGT eekd₁₀kke 18 32016 32031 2198 548662 n/a n/aTGCTTAAAAGTCTCCC eekd₁₀kke 5 32155 32170 2199 548663 n/a n/aGCCCTAGAAGGGCCCA eekd₁₀kke 20 32219 32234 2200 548664 n/a n/aGCGGGTGGTCTTGCAC eekd₁₀kke 38 32245 32260 2201 548665 n/a n/aGCTCCCGGCCATTAGC eekd₁₀kke 8 32312 32327 2202 548666 n/a n/aTCTCCATAGTGAGACG eekd₁₀kke 1 32342 32357 2203 548667 n/a n/aTGGCAAGCTACCTTCT eekd₁₀kke 51 32384 32399 2204 548668 n/a n/aGGGAGCTTTCATGGCT eekd₁₀kke 68 32506 32521 2205 548669 n/a n/aAATGCAGGCCAGCATC eekd₁₀kke 42 32541 32556 2206 548670 n/a n/aGAAAAGCCCTCCGAGC eekd₁₀kke 15 32590 32605 2207 548671 n/a n/aCAACAATCCAAAGCCT eekd₁₀kke 3 32674 32689 2208 548672 n/a n/aCCCCCCAGAAATCCCA eekd₁₀kke 40 32708 32723 2209 548673 n/a n/aGACCTTGCTTCCATGT eekd₁₀kke 40 32753 32768 2210 548674 n/a n/aGAGAGACGGCACCCTG eekd₁₀kke 4 32829 32844 2211 548675 n/a n/aGGGAAGGTAGTGTTAC eekd₁₀kke 8 32898 32913 2212 548676 n/a n/aGTGAATCAGAGCAGTG eekd₁₀kke 63 32963 32978 2213 548677 n/a n/aTCACCTGTGAGTAACC eekd₁₀kke 40 33089 33104 2214 548678 n/a n/aGAGTTACCTTACAAGC eekd₁₀kke 22 33232 33247 2215 548679 n/a n/aTCTCAAGCAGCCTATT eekd₁₀kke 0 33267 33282 2216 548680 n/a n/aGCCCCTCTTAAATAGC eekd₁₀kke 9 33446 33461 2217 548681 n/a n/aGATATCATCATCCCAA eekd₁₀kke 22 33513 33528 2218 548682 n/a n/aGTATCCCCTTTTCTAT eekd₁₀kke 0 33556 33571 2219 548683 n/a n/aAGTATCTCATGTGCCT eekd₁₀kke 46 33581 33596 2220 548684 n/a n/aCAAGACCTTGCTTGCC eekd₁₀kke 24 33658 33673 2221 548685 n/a n/aTAGTCCACTACACAGC eekd₁₀kke 24 33802 33817 2222 548686 n/a n/aACGACAATGGGATTCA eekd₁₀kke 0 33844 33859 2223 548687 n/a n/aGAATCTCCCTGAGTCA eekd₁₀kke 20 33888 33903 2224 548688 n/a n/aTAGAGGGATCCCAGGA eekd₁₀kke 0 34416 34431 2225 548689 n/a n/aCCAGGTGCAGCACGGA eekd₁₀kke 12 34483 34498 2226

Example 4: Dose-Dependent Antisense Inhibition of Human PKK in HepaRG™Cells

Gapmers from the studies described above exhibiting significant in vitroinhibition of PKK mRNA were selected and tested at various doses inHepaRG™ cells. Cells were plated at a density of 20,000 cells per welland transfected using electroporation with 0.12 M, 0.37 M, 1.11 M, 3.33M, and 10.00 M concentrations of antisense oligonucleotide. After atreatment period of approximately 16 hours, RNA was isolated from thecells and PKK mRNA levels were measured by quantitative real-time PCR.Human PKK primer probe set RTS3454 was used to measure mRNA levels. PKKmRNA levels were adjusted according to total RNA content, as measured byRIBOGREEN®. The antisense oligonucleotides were tested in a series ofexperiments that had similar culture conditions. The results for eachexperiment are presented in separate tables shown below. Results arepresented as percent inhibition of PKK, relative to untreated controlcells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented. PKK mRNA levels were significantly reduced in adose-dependent manner in antisense oligonucleotide treated cells.

TABLE 32 0.12 0.37 1.11 3.33 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM)486847 0 34 48 71 87 1.1 530933 15 13 42 67 66 1.7 530959 12 27 53 80 940.9 530965 8 5 63 83 91 0.8 530967 30 36 48 82 91 0.7 530970 1 0 66 7684 1.0 530971 12 40 52 66 70 1.3 530988 0 25 54 86 78 0.9 530992 0 50 6383 80 0.7 531002 6 28 58 82 86 0.9 531004 0 14 25 71 84 2.1 531005 14 2861 73 77 0.9 531022 0 0 32 62 77 2.2 531078 10 27 54 69 92 1.1 531231 2330 76 89 94 0.6

TABLE 33 0.12 0.37 1.11 3.33 10.00 IC₅₀ ISIS No μM μM μM μM μM (μM)531026 23 26 49 75 85 1.0 531055 3 28 64 76 81 0.9 531069 19 39 48 76 830.9 531071 23 37 56 83 83 0.7 531110 14 29 49 76 85 1.1 531121 0 13 4769 79 1.5 531123 14 43 51 71 64 0.9 531172 0 16 37 60 60 2.1 531198 0 3562 76 60 0.8 531231 18 0 36 76 84 2.0 531232 15 26 40 62 76 1.7 53123317 27 50 77 84 1.0 531234 24 21 47 72 82 1.4 531235 27 55 62 84 95 0.4531236 4 28 59 85 93 0.8

Example 5: Dose-Dependent Antisense Inhibition of Human PKK in HepaRG™Cells

Gapmers from the studies described above exhibiting significant in vitroinhibition of PKK mRNA were selected and tested at various doses inHepaRG™ cells. Cells were plated at a density of 20,000 cells per welland transfected using electroporation with 0.19 μM, 0.56 μM, 1.67 μM,and 5.00 μM concentrations of antisense oligonucleotide. After atreatment period of approximately 16 hours, RNA was isolated from thecells and PKK mRNA levels were measured by quantitative real-time PCR.Human PKK primer probe set RTS3454 was used to measure mRNA levels. PKKmRNA levels were adjusted according to total RNA content, as measured byRIBOGREEN. The antisense oligonucleotides were tested in a series ofexperiments that had similar culture conditions. The results for eachexperiment are presented in separate tables shown below. Results arepresented as percent inhibition of PKK, relative to untreated controlcells. ‘n/a’ indicates that there was no measurement done for thatparticular antisense oligonucleotide for that particular dose.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented. PKK mRNA levels were significantly reduced in adose-dependent manner in antisense oligonucleotide treated cells.

TABLE 34 0.19 0.56 1.67 5.00 IC₅₀ ISIS No μM μM μM μM (μM) 531231 32 3073 89 0.5 546158 5 45 79 83 0.7 546188 36 55 81 83 0.4 546253 1 13 46 811.7 546254 51 66 80 91 0.2 546343 28 64 87 87 0.4 546825 46 73 86 88 0.2546827 32 70 84 90 0.3 546828 39 58 87 93 0.3 546829 3 30 73 88 1.0546846 36 45 71 82 0.5 547413 0 0 41 83 2.2 547423 37 50 92 90 0.4547445 41 75 82 88 0.2 547456 12 67 66 80 1.0 547464 21 52 67 97 0.6547564 51 48 82 90 0.2 547587 20 62 84 86 0.5 548758 41 47 82 94 0.4

TABLE 35 0.19 0.56 1.67 5.00 IC₅₀ ISIS No μM μM μM μM (μM) 531231 25 3484 92 0.7 546190 33 65 86 n/a 0.4 546208 16 45 79 91 0.7 546216 62 69 8888 0.1 546255 32 35 78 87 0.5 546268 56 50 82 93 0.1 546301 25 50 53 870.8 546849 23 35 83 91 0.7 546852 19 40 78 85 0.8 546889 23 54 78 91 0.6546916 43 71 79 89 0.2 546967 20 39 76 71 0.7 547273 44 69 87 87 0.2547276 35 44 71 77 0.6 547335 8 52 85 92 0.7 547340 46 79 88 n/a 0.2547602 18 53 92 87 0.5 547647 1 70 72 n/a 0.8 547694 0 29 67 90 1.2

TABLE 36 0.19 0.56 1.67 5.00 IC₅₀ ISIS No μM μM μM μM (μM) 531231 58 6477 98 0.1 546247 0 29 71 88 1.1 546251 31 60 99 89 0.5 546753 28 47 8396 0.5 546826 17 40 87 97 0.7 546833 8 33 74 94 0.9 546854 23 39 83 940.6 546894 15 47 50 93 0.9 546897 40 56 71 95 0.4 546903 15 37 74 98 0.8546986 31 49 77 89 0.5 547293 53 57 80 86 0.2 547298 32 61 74 90 0.4547364 38 47 54 89 0.6 547373 20 7 49 86 1.1 547426 19 50 84 93 0.6547454 19 40 58 92 0.9 547617 52 66 77 93 0.2 548770 26 54 77 91 0.5

TABLE 37 0.19 0.56 1.67 5.00 IC₅₀ ISIS No μM μM μM μM (μM) 531231 34 4772 n/a 0.5 546214 0 0 68 85 1.3 546304 0 6 51 71 2.1 546739 35 55 57 790.6 546832 19 38 70 95 0.8 546847 39 57 75 89 0.4 546855 18 7 30 82 2.2546877 0 19 75 80 1.3 546939 1 66 86 90 0.6 547349 0 8 66 76 1.6 5473608 27 76 76 0.8 547368 0 0 31 80 2.5 547483 0 9 49 71 2.1 547575 0 34 8293 1.1 547618 0 0 73 98 1.3 547622 0 47 79 90 0.9 547637 10 21 36 82 1.8547731 0 0 17 56 5.0 548752 0 0 51 90 1.9

TABLE 38 0.19 0.56 1.67 5.00 IC₅₀ ISIS No μM μM μM μM (μM) 531231 21 4567 96 0.7 546195 34 51 79 92 0.5 546198 7 3 45 92 1.3 546287 0 15 39 891.7 546358 0 19 71 80 1.3 546403 0 20 37 41 >5.0 546410 13 43 52 75 1.2546412 0 1 61 62 2.3 546429 6 10 44 69 2.3 546834 1 0 30 83 2.3 547006 00 54 77 1.5 547294 28 59 87 86 0.4 547337 23 41 55 79 1.0 547514 18 8 5180 1.9 547584 26 34 76 86 0.7 547585 42 57 70 95 0.4 547615 20 26 41 841.4 547636 0 24 79 94 1.1 548744 14 35 63 83 1.0

TABLE 39 0.19 0.56 1.67 5.00 IC₅₀ ISIS No μM μM μM μM (μM) 531231 21 3990 97 0.6 546232 49 50 94 97 0.2 546248 25 66 87 93 0.4 546835 9 35 6893 0.9 546848 0 18 91 97 1.0 546853 47 64 84 n/a 0.2 546870 35 42 80 950.5 546872 32 33 82 94 0.4 546876 0 50 85 95 0.8 547275 34 66 82 95 0.3547341 36 58 91 95 0.3 547366 0 45 68 91 1.2 547453 25 40 54 92 0.8547457 41 65 80 85 0.3 547616 26 50 72 89 0.6 547632 44 47 81 97 0.6547633 12 46 78 n/a 0.7 547718 36 12 69 74 1.6 548757 18 49 82 93 0.6

TABLE 40 0.19 0.56 1.67 5.00 IC₅₀ ISIS No μM μM μM μM (μM) 531231 6 3874 95 0.8 546291 22 32 34 72 2.0 546310 0 36 56 80 1.3 546896 0 45 82 970.8 546980 0 18 29 80 2.2 547009 0 9 21 63 3.6 547019 0 6 54 86 1.6547277 2 32 34 62 2.8 547288 0 0 0 38 >5.0 547374 0 15 24 44 >5.0 5474930 26 64 77 1.3 547520 0 25 66 64 1.1 547712 0 5 21 62 3.8 547722 0 15 3273 2.4 547728 0 2 16 61 4.4 547780 0 10 36 55 3.9 548743 25 57 73 88 0.5548753 0 23 49 84 1.5 548756 0 4 16 86 >5.0

TABLE 41 0.19 0.56 1.67 5.00 IC₅₀ ISIS No μM μM μM μM (μM) 531231 25 5589 97 0.5 546188 27 69 88 97 0.4 546216 23 78 95 98 <0.2 546254 40 63 8495 0.3 546268 0 71 92 92 0.5 546343 37 32 83 95 0.4 546825 38 82 n/a 990.2 546827 23 74 98 96 0.4 546828 0 64 89 97 0.2 546846 26 49 85 n/a 0.5546967 22 45 74 92 0.7 547273 0 60 82 83 0.6 547340 34 84 96 n/a 0.3547423 78 92 n/a n/a <0.2 547445 80 87 98 91 <0.2 547564 46 66 90 97 0.2547587 38 64 91 97 0.3 547602 1 9 52 93 1.4 548758 0 72 79 n/a 0.6

TABLE 42 0.19 0.56 1.67 5.00 IC₅₀ ISIS No μM μM μM μM (μM) 531231 7 3956 97 1.0 546190 21 34 76 98 0.7 546208 5 33 70 97 0.9 546251 19 45 9197 0.6 546255 5 39 82 96 0.8 546739 4 62 84 86 0.6 546753 17 31 70 910.9 546849 13 45 84 98 0.7 546889 25 9 73 92 1.4 546897 16 17 69 97 0.8546916 0 27 73 97 1.0 546986 7 28 69 86 1.1 547276 6 3 53 68 2.2 5472930 45 65 70 1.3 547298 0 12 67 87 1.7 547335 0 13 73 95 1.3 547426 18 3580 95 0.7 547617 17 37 79 98 0.7 548770 9 0 61 92 1.7

TABLE 43 0.19 0.56 1.67 5.00 IC₅₀ ISIS No μM μM μM μM (μM) 531231 6 5668 97 0.8 546195 0 27 91 94 0.9 546232 0 74 95 96 0.2 546248 0 59 73 890.8 546832 36 49 85 97 0.4 546847 14 44 83 95 0.7 546853 4 49 74 92 0.8546870 36 34 61 91 1.0 546872 42 13 59 99 1.4 546896 35 60 83 n/a 0.4546939 16 71 96 95 0.4 547275 56 16 80 86 1.2 547294 4 70 84 91 0.6547341 45 44 81 95 0.6 547457 33 42 70 83 0.6 547584 0 21 64 92 1.3547585 0 46 89 93 0.8 547632 0 0 63 91 1.6 548743 22 47 74 96 0.6

Example 6: Dose-Dependent Antisense Inhibition of Human PKK in HepaRG™Cells

Gapmers from the studies described above exhibiting significant in vitroinhibition of PKK mRNA were selected and tested at various doses inHepaRG™ cells. Cells were plated at a density of 20,000 cells per welland transfected using electroporation with 0.11 M, 0.33 M, 1.00 M, and3.00 M concentrations of antisense oligonucleotide. After a treatmentperiod of approximately 16 hours, RNA was isolated from the cells andPKK mRNA levels were measured by quantitative real-time PCR. Human PKKprimer probe set RTS3454 was used to measure mRNA levels. PKK mRNAlevels were adjusted according to total RNA content, as measured byRIBOGREEN®. The antisense oligonucleotides were tested in a series ofexperiments that had similar culture conditions. The results for eachexperiment are presented in separate tables shown below. Results arepresented as percent inhibition of PKK, relative to untreated controlcells. ‘n/a’ indicates that there was no measurement done for thatparticular antisense oligonucleotide for that particular dose.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented. PKK mRNA levels were significantly reduced in adose-dependent manner in antisense oligonucleotide treated cells.

TABLE 44 0.11 0.33 1.00 3.00 IC₅₀ ISIS No μM μM μM μM (μM) 547747 24 2981 89 0.4 547769 12 17 80 96 0.6 547824 45 73 78 n/a 0.1 547835 44 27 5379 0.9 547843 0 52 80 91 0.4 547857 36 66 77 93 0.2 547870 0 44 80 970.6 547943 33 70 87 90 0.2 547946 0 47 74 n/a 0.5 547947 24 58 81 93 0.3547998 55 73 91 91 0.1 548004 24 47 80 92 0.3 548010 0 11 49 64 1.4548047 50 62 76 95 0.1 548147 59 94 80 n/a 0.0 548338 41 58 79 95 0.2548348 19 46 67 91 0.4 548409 21 60 90 93 0.3 548557 5 47 82 95 0.4

TABLE 45 0.11 0.33 1.00 3.00 IC₅₀ ISIS No μM μM μM μM (μM) 547747 8 6190 92 0.3 547807 26 71 61 94 0.4 547922 67 75 81 92 0.0 547927 56 64 9288 0.1 547948 60 80 88 97 0.0 547979 56 58 94 97 0.1 548005 53 49 71 950.4 548024 28 57 84 82 0.3 548043 14 60 90 92 0.3 548055 43 57 50 88 0.3548106 53 54 82 94 0.1 548109 50 92 79 85 0.1 548155 49 50 70 81 0.3548180 11 59 71 88 0.4 548278 3 59 78 93 0.4 548343 61 67 88 92 0.0548558 53 61 78 95 0.1 548570 20 40 70 94 0.4 548583 43 46 93 88 0.2

TABLE 46 0.11 0.33 1.00 3.00 IC₅₀ ISIS No μM μM μM μM (μM) 547747 3 4472 90 0.5 547849 36 52 67 n/a 0.3 547851 16 46 83 n/a 0.4 547859 29 5683 78 0.3 547862 26 71 69 n/a 0.3 547877 29 66 83 n/a 0.2 547942 25 5191 n/a 0.3 547997 39 68 n/a 82 0.2 548046 7 35 64 77 0.7 548048 49 66 8692 0.1 548061 26 61 59 n/a 0.4 548070 26 35 48 63 1.1 548125 33 50 81 730.3 548195 5 23 61 76 0.8 548265 47 69 78 67 0.1 548410 31 58 85 82 0.2548424 17 67 86 72 0.3 548425 41 57 68 80 0.2 548547 30 41 76 90 0.4

TABLE 47 0.11 0.33 1.00 3.00 IC₅₀ ISIS No μM μM μM μM (μM) 547747 16 5985 96 0.3 547808 19 22 48 71 1.1 547861 7 40 75 84 0.5 548069 6 0 27 661.9 548128 14 29 49 66 1.1 548170 0 8 26 65 2.0 548174 20 18 29 62 2.0548197 33 37 51 75 0.8 548201 0 7 70 85 0.8 548217 22 24 54 71 0.9548220 0 0 0 6 >3 548247 16 50 62 82 0.5 548422 0 32 71 93 0.7 548479 252 82 97 0.4 548486 20 48 77 92 0.4 548521 21 0 3 1 >3 548655 0 0 833 >3 548667 0 37 73 86 0.7 548668 10 30 61 84 0.7

Example 7: Efficacy of Antisense Oligonucleotides Targeting Human PKK inTransgenic Mice

Transgenic mice containing a 37,390 base pair fragment of the humanKLKB1 gene sequence (chromosome 4: position 187148672-187179625,accession no: NC_000004.11) were treated with ISIS antisenseoligonucleotides selected from studies described above, which wereevaluated for efficacy in this model.

Treatment

Groups of transgenic mice were injected subcutaneously twice a week for3 weeks with 2.5 mg/kg/week, 5.0 mg/kg/week, 10 mg/kg/week or 20mg/kg/week of ISIS 546232, ISIS 546251, ISIS 546254, ISIS 546343, ISIS546828, ISIS 547455, ISIS 547457, ISIS 547927, and ISIS 548048. Onegroup of transgenic mice was injected subcutaneously twice a week for 3weeks with PBS. Mice were euthanized 48 hours after the last dose, andorgans and plasma were harvested for further analysis.

RNA Analysis

To evaluate the effect of ISIS oligonucleotides on target reduction, RNAwas extracted from liver tissue for real-time PCR analysis of human PKK.Results are presented as percent inhibition of PKK mRNA, relative to PBScontrol. As shown in Table 48, treatment with ISIS antisenseoligonucleotides resulted in significant reduction of PKK mRNA incomparison to the PBS control.

TABLE 48 Percent Inhibition of PKK mRNA in the transgenic mice liverrelative to the PBS control % ISIS No Dose inhibition 547927 20 71 10 935 52 2.5 35 547455 20 62 10 45 5 69 2.5 0 546232 20 84 10 30 5 53 2.5 57546254 20 83 10 84 5 55 2.5 31 546343 20 86 10 66 5 n/a 2.5 46 548048 2080 10 72 5 77 2.5 7 546828 20 83 10 32 5 62 2.5 77 546251 20 79 10 66 551 2.5 13 547457 20 62 10 45 5 69 2.5 0

Protein Analysis

Plasma PKK protein levels were evaluated in all groups. Results arepresented as percent inhibition of PKK protein, relative to PBS control.As shown in Table 49, treatment with ISIS antisense oligonucleotidesresulted in significant reduction of PKK protein levels in comparison tothe PBS control.

TABLE 49 Percent reduction of PKK protein levels in the transgenic micerelative to the PBS control % ISIS No Dose inhibition 547927 20 80 10n/a 5 21 2.5 25 547455 20 78 10 32 5 0 2.5 0 546232 20 79 10 33 5 6 2.50 546254 20 76 10 51 5 36 2.5 0 546343 20 79 10 38 5 n/a 2.5 0 548048 2098 10 89 5 70 2.5 23 546828 20 93 10 36 5 25 2.5 0 546251 20 69 10 52 530 2.5 22 547457 20 60 10 31 5 4 2.5 0

Example 8: Effect of ISIS Antisense Oligonucleotides Targeting Human PKKin Cynomolgus Monkeys

Cynomolgus monkeys were treated with ISIS antisense oligonucleotidesselected from studies described above. Antisense oligonucleotideefficacy and tolerability were evaluated. The human antisenseoligonucleotides tested are cross-reactive with the rhesus genomicsequence (GENBANK Accession No. NW_001118167.1 truncated fromnucleotides 2358000 to 2391000 and designated herein as SEQ ID NO: 18).The target start site of each oligonucleotide to SEQ ID NO: 18 ispresented in Table 50. ‘Mismatches’ indicates that the number ofnucleotides by which the oligonucleotide is mismatched to the rhesussequence. The greater the complementarity between the humanoligonucleotide and the rhesus monkey sequence, the more likely thehuman oligonucleotide can cross-react with the rhesus monkey sequence.‘n/a’ indicates that the oligonucleotide is has more than 3 mismatcheswith the rhesus gene sequence.

TABLE 50 Antisense oligonucleotides complementary to SEQ ID NO: 18Target SEQ ID ISIS No Start Site Mismatches Sequence Chemistry NO.547927 22059 1 ATGGTCCGACACACAA Deoxy, MOE and cEt 1548 546232 n/a n/aAGGAACTTGGTGTGCCACTT 5-10-5 MOE 526 547455 27391 0 ATATCATGATTCCCTTCTGA5-10-5 MOE 657 546254 23858 1 TGCAAGTCTCTTGGCAAACA 5-10-5 MOE 570 54634330532 0 CCCCCTTCTTTATAGCCAGC 5-10-5 MOE 705 548048 27397 0CGATATCATGATTCCC Deoxy, MOE and cEt 1666 546828 13632 1ACAGTATCACTGTACTAGTT 5-10-5 MOE 904 546251 23846 0 GGCAAACATTCACTCCTTTA5-10-5 MOE 566 547457 27397 0 AAGGCGATATCATGATTCCC 5-10-5 MOE 660

Treatment

Prior to the study, the monkeys were kept in quarantine for a 30-dayperiod, during which the animals were observed daily for general health.The monkeys were 2-4 years old and weighed between 2 and 4 kg. Tengroups of four randomly assigned male cynomolgus monkeys each wereinjected subcutaneously with ISIS oligonucleotide or PBS. PBS solutionor ISIS oligonucleotides at a dose of 40 mg/kg were administeredinitially with a loading regimen consisting of four doses on the firstweek of the study (days 1, 3, 5, and 7), followed by a maintenanceregimen consisting of once weekly administration starting on Day 14(weeks 2 to 13). Subcutaneous injections were performed in clock-wiserotations at 4 sites on the back; one site per dose. The injection siteswere delineated by tattoo, while sedated using ketamine, and wereseparated by a minimum of 3 cm.

During the study period, the monkeys were observed a minimum of oncedaily for signs of illness or distress. Any animal experiencing morethan momentary or slight pain or distress due to the treatment, injuryor illness was promptly reported to the responsible veterinarian and theStudy Director. Any animal in poor health or in a possible moribundcondition was identified for further monitoring and possible euthanasia.For example, two monkeys treated with ISIS 547445 were euthanized due tosubdued behavior, lateral position, lack of response to stimuli anddecreased respiration. The protocols described in the Example wereapproved by the Institutional Animal Care and Use Committee (IACUC).

Target Reduction RNA Analysis

On day 87 or 88, 48 hours after the final dose, RNA was extracted fromliver tissue for real-time PCR analysis of PKK using primer probe setRTS3455 (forward sequence CCTGTGTGGAGGGTCACTCA, designated herein as SEQID NO: 23; reverse sequence CCACTATAGATGCGCCAAACATC, designated hereinas SEQ ID NO: 24; probe sequence CCCACTGCTTTGATGGGCTTCCC, designatedherein as SEQ ID NO: 25). The results were normalized to thehousekeeping gene, Cyclophilin. Results are presented as percentinhibition of PKK mRNA, relative to PBS control. As shown in Table 51,treatment with ISIS antisense oligonucleotides resulted in significantreduction of PKK mRNA in comparison to the PBS control.

TABLE 51 Percent Inhibition of PKK mRNA in the cynomolgus monkey liverrelative to the PBS control % ISIS No inhibition 546232 88 546251 90546254 88 546343 74 546828 45 547455 90 547457 89 547927 54 548048 95

Protein Analysis

Approximately 0.9 mL of blood was collected each time from all availableanimals at pre-dose, day 17, day 31, day 45, day 59, and day 73, andplaced in tubes containing 3.2% sodium citrate. The tubes werecentrifuged (3000 rpm for 10 min at room temperature) to obtain plasma.PKK protein levels were measured in the plasma by ELISA. The results arepresented in Table 52, expressed as percentage inhibition compared tothe PBS control levels. The results indicate that ISIS oligonucleotidessignificantly reduced PKK protein levels.

TABLE 52 PKK protein level reduction (%) in the cynomolgus monkey plasmarelative to control levels Day 17 Day 31 Day 45 Day 59 Day 73 ISIS546232 53 58 72 75 70 ISIS 546251 71 75 75 81 77 ISIS 546254 38 51 63 7473 ISIS 546343 56 74 69 70 70 ISIS 546828 0 8 23 39 39 ISIS 547455 26 3343 58 58 ISIS 547457 68 75 79 76 80 ISIS 547927 8 0 15 10 18 ISIS 54804890 93 95 95 95

Tolerability Studies Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, themonkeys were fasted overnight. Approximately, 1.5 mL of blood sampleswere collected from all the study groups. Blood was collected in tubeswithout anticoagulant for serum separation. The tubes were kept at roomtemperature for a minimum of 90 min and then centrifuged at 3,000 rpmfor 10 min. Levels of various liver function markers were measured usinga Toshiba 120FR NEO chemistry analyzer (Toshiba Co., Japan). The resultsare presented in Table 53 and indicate that antisense oligonucleotideshad no effect on liver function outside the expected range for antisenseoligonucleotides.

TABLE 53 Liver function markers in cynomolgus monkey plasma Albumin ASTALT (g/dL) (IU/L) (IU/L) PBS 4.2 48 60 ISIS 546232 4.1 63 140 ISIS546251 3.7 51 58 ISIS 546254 3.8 68 54 ISIS 546343 4.3 49 76 ISIS 5468283.7 75 67 ISIS 547455 3.8 56 61 ISIS 547457 4.0 54 52 ISIS 547927 4.2 5961 ISIS 548048 4.2 44 47

Hematology

To evaluate any effect of ISIS oligonucleotides in cynomolgus monkeys onhematologic parameters, blood samples of approximately 1.2 mL of bloodwas collected pre-dose and on day 87 or day 88 from each of theavailable study animals in tubes containing K₂-EDTA. Samples wereanalyzed for red blood cell (RBC) count, white blood cells (WBC) count,platelet count, hemoglobin content and hematocrit, using an ADVIA2120ihematology analyzer (SIEMENS, USA). The data is presented in Table 54.

The data indicate treatment with most of the oligonucleotides did notcause any changes in hematologic parameters outside the expected rangefor antisense oligonucleotides at this dose.

TABLE 54 Hematological parameters in cynomolgus monkeys RBC PlateletsWBC Hemoglobin HCT (×10⁶/μL) (×10^(3/)μL) (×10³/μL) (g/dL) (%) PBS 5.4458 13 13.1 43 ISIS 546232 5.4 391 11 12.9 42 ISIS 546251 5.7 419 8 12.943 ISIS 546254 5.3 436 11 12.4 41 ISIS 546343 5.5 373 14 12.6 42 ISIS546828 6.0 408 11 12.9 43 ISIS 547455 4.5 448 13 10.2 34 ISIS 547457 6.4367 10 13.8 45 ISIS 547927 5.2 461 45 12.5 41 ISIS 548048 5.9 393 1113.4 44

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, themonkeys were fasted overnight. Approximately, 1.5 mL of blood sampleswere collected from all the study groups. Blood was collected in tubeswithout anticoagulant for serum separation. The tubes were kept at roomtemperature for a minimum of 90 min and then centrifuged at 3,000 rpmfor 10 min. Levels of BUN and creatinine were measured using a Toshiba120FR NEO chemistry analyzer (Toshiba Co., Japan). Results are presentedin Table 55, expressed in mg/dL. The plasma chemistry data indicate thatmost of the ISIS oligonucleotides did not have any effect on the kidneyfunction outside the expected range for antisense oligonucleotides.Specifically, treatment with ISIS 546254 was well tolerated in terms ofthe kidney function of the monkeys.

Kidney function was also assessed by urinalysis. Fresh urine from allanimals was collected using a clean cage pan on wet ice. Food wasremoved overnight the day before fresh urine collection was done butwater was supplied. The total protein and creatinine levels weremeasured using a Toshiba 120FR NEO automated chemistry analyzer (ToshibaCo., Japan) and the protein to creatinine ratio was calculated. Theresults are presented in Table 56.

TABLE 55 Plasma BUN and creatinine levels (mg/dL) in cynomolgus monkeysBUN Creatinine PBS 22.8 0.9 ISIS 546232 22.7 1.0 ISIS 546251 25.4 1.1ISIS 546254 25.7 0.9 ISIS 546343 26.2 1.0 ISIS 546828 24.7 0.9 ISIS547455 29.4 0.9 ISIS 547457 24.3 1.0 ISIS 547927 22.3 1.0 ISIS 54804821.9 0.9

TABLE 56 Urine protein/creatinine ratio in cynomolgus monkeys Ratio ISIS546232 0.03 ISIS 546251 0.12 ISIS 546254 0.04 ISIS 546343 0.01 ISIS546828 0.03 ISIS 547455 0.70 ISIS 547457 0.03 ISIS 547927 0.04 ISIS548048 0.03 PBS 0.06

C-Reactive Protein Level Analysis

To evaluate any inflammatory effect of ISIS oligonucleotides incynomolgus monkeys, the monkeys were fasted overnight. Approximately,1.5 mL of blood samples were collected from all the study groups. Bloodwas collected in tubes without anticoagulant for serum separation. Thetubes were kept at room temperature for a minimum of 90 min and thencentrifuged at 3,000 rpm for 10 min. C-reactive protein (CRP), which issynthesized in the liver and which serves as a marker of inflammation,was measured using a Toshiba 120FR NEO chemistry analyzer (Toshiba Co.,Japan). Complement C3 was also measured similarly, and the data ispresented as a percentage of baseline values. The results are presentedin Table 57 and indicate that treatment with ISIS oligonucleotides didnot cause any inflammation in monkeys.

TABLE 57 C-reactive protein and C3 levels in cynomolgus monkey plasmaCRP C3 (mg/dL) (% of baseline) PBS 0.2 73 ISIS 546232 0.5 50 ISIS 5462510.7 62 ISIS 546254 0.8 61 ISIS 546343 0.2 60 ISIS 546828 0.6 56 ISIS547455 1.9 64 ISIS 547457 0.3 53 ISIS 547927 0.2 73 ISIS 548048 0.2 69

Example 9: Antisense Inhibition of Murine PKK mRNA in Mouse PrimaryHepatocytes

Antisense oligonucleotides targeting a murine PKK nucleic acid weredesigned and tested for their effects on PKK mRNA in vitro. Culturedmouse primary hepatocytes at a density of 10,000 cells per well weretransfected using Cytofectin reagent with 12.5 nM, 25.0 nM, 50.0 nM,100.0 nM, and 200.0 nM of antisense oligonucleotide. After a treatmentperiod of approximately 24 hours, RNA was isolated from the cells andmouse PKK mRNA levels were measured by quantitative real-time PCR usingthe murine primer probe set RTS3313 (forward sequenceTGCCTGCTGTTCAGCTTTCTC, designated herein as SEQ ID NO: 2228; reversesequence TGGCAAAGTCCCTGTAATGCT, designated herein as SEQ ID NO: 2229;probe sequence CGTGACTCCACCCAAAGAGACAAATAAACG, designated herein as SEQID NO: 2230). PKK mRNA levels were adjusted according to total RNAcontent, as measured by RIBOGREEN.

The chimeric antisense oligonucleotides were designed as 5-10-5 MOEgapmers. The gapmers are 20 nucleotides in length, wherein the centralgap segment is comprised of ten 2′-deoxynucleosides and is flanked onboth sides (in the 5′ and 3′ directions) by wings comprising 5nucleosides each. Each nucleoside in the 5′ wing segment and eachnucleoside in the 3′ wing segment has a 2′-O-methoxyethyl modification.The internucleoside linkages throughout each gapmer are phosphorothioatelinkages. All cytosine residues throughout each gapmer are5-methylcytosines. Results demonstrate that PKK mRNA levels weresignificantly reduced in a dose dependent manner.

In one specific example, ISIS 482584 (GGCATATTGGTTTTTGGAAT; SEQ ID NO:2244) reduced PKK mRNA in a dose dependent manner yielding a halfmaximal inhibitory concentration (IC₅₀) of 84 nM (see Table 58). ISIS482584 is targeted to SEQ ID NO: 11 (GENBANK Accession No. NM_008455.2)and has a target start site of 1586 and a target stop site of 1605.“Target start site” indicates the 5′-most nucleotide to which the gapmeris targeted. “Target stop site” indicates the 3′-most nucleotide towhich the gapmer is targeted.

TABLE 58 Dose-dependent inhibition of mouse PKK mRNA levels by ISIS482584 % Dose inhibition 12.5 nM 0 25.0 nM 47 50.0 nM 27 100.0 nM 60200.0 nM 82

Example 10: Antisense Inhibition of PKK mRNA in BALB/c Mice

ISIS 482584 was tested for its effect on murine PKK mRNA in vivo.

Treatment

Six groups of male BALB/c mice each were treated with 2.5 mg/kg, 5.0mg/kg, 10.0 mg/kg, 20.0 mg/kg, 40.0 mg/kg, or 80.0 mg/kg of ISIS 482584,administered subcutaneously twice a week for 3 weeks (weekly doses of5.0 mg/kg, 10.0 mg/kg, 20.0 mg/kg, 40.0 mg/kg, 80.0 mg/kg, or 160.0mg/kg). A control group of BALB/c mice was treated with PBS,administered subcutaneously twice a week for 3 weeks. Two days after thelast dose of antisense oligonucleotide or PBS, mice from all groups wereanesthetized with 150 mg/kg ketamine mixed with 10 mg/kg xylazine,administered by intraperitoneal injection. Liver was collected for RNAanalysis.

RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of PKK.PKK mRNA levels were measured using the murine primer probe set (forwardsequence ACAAGTGCATTTTACAGACCAGAGTAC, designated herein as SEQ ID NO:2231; reverse sequence GGTTGTCCGCTGACTTTATGCT, designated herein as SEQID NO: 2232; probe sequence AAGCACAGTGCAAGCGGAACACCC, designated hereinas SEQ ID NO: 2233). Results are presented as percent inhibition of PKK,relative to PBS control. As shown in Table 59, treatment with ISIS482584 resulted in significant dose-dependent reduction of PKK mRNA incomparison to the PBS control.

TABLE 59 Dose-dependent reduction of PKK mRNA in BALB/c mice liver Dose% (mg/kg/wk) inhibition 5 3 10 42 20 68 40 85 80 91 160 94

Protein Analysis

Plasma was collected in tubes containing sodium citrate as ananticoagulant. The samples were run on a 4-12% gradientSDS-polyacrylamide gel (Invitrogen), followed by immunoblotting withmurine PKK antibody (R&D Systems). Blots were incubated with secondaryfluorophore-labeled antibodies (LI-COR) and imaged in an Odyssey Imager(LI-COR). Results are presented as percent inhibition of PKK, relativeto PBS control. As shown in Table 60, treatment with ISIS 482584resulted in significant dose-dependent reduction of PKK plasma proteinin comparison to the PBS control.

TABLE 60 Dose-dependent reduction of PKK protein in BALB/c mice plasmaDose % (mg/kg/wk) inhibition 5 5 10 24 20 47 40 76 80 81 160 n.d. n.d. =no data

Example 11: In Vivo Effect of Antisense Inhibition of Murine PKK in anAngioedema Mouse Model

Hereditary angioedema (HAE) is characterized by local swelling andincrease in vascular permeability in subcutaneous tissues (Morgan, B. P.N. Engl. J. Med. 363: 581-83, 2010). It is caused by a deficiency of theC1 inhibitor, a protein of the complement system. Two mouse models wereused in this study including an established mouse model of C1-INHdeficiency and a captopril-induced edema model, both of which causevascular permeability, a hallmark of HAE. Reversal of vascularpermeability is accompanied by increased plasma levels of high molecularweight kininogen (HMWK).

In the first model, angioedema was induced by treatment with Captopril,a known antihypertensive agent, which increases vascular permeability inmice and replicates the pathology of hereditary angioedema.

In the second model, angioedema was induced by treatment with ISIS461756, an antisense oligonucleotide which targets murine C1 inhibitormRNA, which increases vascular permeability in mice and replicates thepathology of hereditary angioedema. ISIS 461756 (SEQ ID NO: 2245;AAAGTGGTTGATACCCTGGG) is a 5-10-5 MOE gapmer targeting nucleosides1730-1749 of NM_009776.3 (SEQ ID NO: 2243).

The effect of HOE-140 and ISIS 482584, an antisense oligonucleotideinhibitor of PKK, were evaluated in the Captopril and ISIS461756-induced mouse models of vascular permeability. Some of the murinegroups were treated with HOE-140, a selective antagonist of thebradykinin B2 receptor, which blocks vasodilation and vascularpermeability (Cruden and Newby, Expert Opin. Pharmacol. 9: 2383-90,2008). Other mice were treated with ISIS 482584, which inhibits PKK mRNAexpression. The effect of treatment with HOE-140 was compared with theeffect of treatment with ISIS 482584.

Treatment

The various treatment groups for this assay are presented in Table 61.

Group 1 consisted of 4 C57BL/6J-Tyrc-2J mice treated with PBSadministered subcutaneously twice a week for 4 weeks. No other treatmentwas administered to Group 1 which served as a control group to measurethe basal level of vascular permeability.

Group 2 consisted of 8 C57BL/6J-Tyrc-2J mice treated with PBSadministered subcutaneously twice a week for 4 weeks. At the end of thetreatment, the mice were intraperitoneally administered 20 μg ofcaptopril. Group 2 served as a PBS control group for captopril-inducedvascular permeability.

Group 3 consisted of 8 C57BL/6J-Tyrc-2J mice treated with PBSadministered subcutaneously twice a week for 4 weeks. On day 14, themice were treated with 50 mg/kg of the antisense oligonucleotidetargeting C1 inhibitor, ISIS 461756, administered subcutaneously twice aweek for 2 weeks. At the end of the treatment period, the mice wereintraperitoneally administered 20 μg of captopril. Group 3 served as aPBS control group for captopril and ISIS 461756-induced vascularpermeability.

Group 4 consisted of 8 C57BL/6J-Tyrc-2J mice treated with PBSadministered subcutaneously twice a week for 4 weeks. On day 14, themice were treated with 50 mg/kg of the antisense oligonucleotidetargeting C1 inhibitor, ISIS 461756, administered subcutaneously twice aweek for 2 weeks. At the end of the treatment period, the mice wereintraperitoneally administered 20 μg of captopril. The mice were thenalso intraperitoneally administered 30 μg of HOE-140. Group 4 served asa positive control for inhibition of vascular permeability with HOE-140.

Group 5 consisted of 8 C57BL/6J-Tyrc-2J mice treated with 40 mg/kg ofcontrol oligonucleotide ISIS 141923, a 5-10-5 MOE gapmer with no knownmurine target, (CCTTCCCTGAAGGTTCCTCC; SEQ ID NO: 2246) administeredsubcutaneously twice a week for 4 weeks. On day 14, the mice weretreated with 50 mg/kg of the antisense oligonucleotide targeting C1inhibitor, ISIS 461756, administered subcutaneously twice a week for 2weeks. At the end of the treatment period, the mice wereintraperitoneally administered 20 μg of captopril. Group 5 served as acontrol group for captopril and ISIS 461756-induced vascularpermeability.

Group 6 consisted of 8 C57BL/6J-Tyrc-2J mice and was treated with 40mg/kg of ISIS 482584 administered subcutaneously twice a week for 4weeks. At the end of the treatment period, the mice wereintraperitoneally administered 20 μg of captopril. Group 6 served as theexperimental treatment group for examining the effect of PKK ASO oncaptopril-induced vascular permeability.

Group 7 consisted of 8 C57BL/6J-Tyrc-2J mice treated with 40 mg/kg ofISIS 482584 administered subcutaneously twice a week for 4 weeks. On day14, the mice were treated with 50 mg/kg of the antisense oligonucleotidetargeting C1 inhibitor, ISIS 461756, administered subcutaneously twice aweek for 2 weeks. At the end of the treatment period, the mice wereintraperitoneally administered 20 μg of captopril. Group 7 served as theexperimental treatment group for examining the effect of PKK ASO oncaptopril and ISIS 461756-induced vascular permeability.

All the groups were then injected with 30 mg/kg of Evans Blue solutioninto the tail vein. The mice were sacrificed 30 min after the Evans Bluesolution administration and colons, feet, ears, and intestines wereharvested. Blood samples were taken through cardiac puncture.

TABLE 61 Treatment groups Group No. Treatment Captopril ISIS 461756HOE-140 1. (N = 4) PBS No No No 2. (N = 8) PBS Yes No No 3. (N = 8) PBSYes Yes No 4. (N = 8) PBS Yes Yes Yes 5. (N = 8) ISIS 141923 Yes Yes No6. (N = 8) ISIS 482584 Yes No No 7. (N = 8) ISIS 482584 Yes Yes No

Quantification of Vascular Permeability

The harvested tissues from the feet, colon, ears, and intestines wereplaced separately in formamide solution overnight to leach out the EvansBlue dye. The formamide solution containing ear and feet tissue washeated to 55° C. and left overnight. The color intensity of thedye-infused formamide solution was then measured at OD_(600 nm), and ispresented in Table 62. Mice displaying any manifestation of angioedematake up more dye and, therefore, demonstrate high OD values.

As presented in Table 62, treatment with ISIS 482584 prevents vascularpermeability in mice treated with captopril (Group 6) and in micetreated with captopril and ISIS 461756 (Group 7) compared to therespective PBS control groups (Groups 2 and 3). Measures of vascularpermeability in mice of Groups 6 and 7 were also reduced in most of thetissues in comparison to the mice treated with the controloligonucleotide, ISIS 141923 (Group 5), where vascular permeability wasinduced with captopril and ISIS 461756. Measures of vascularpermeability in the colon and feet tissues of both the treatment groups(Groups 6 and 7) were comparable to basal levels, as observed in micetreated with only PBS (Group 1). Reduction in vascular permeability inmice treated with ISIS 482584 was comparable to that seen in micetreated with the bradykinin 2 receptor antagonist, HOE140, which servedas a positive control in this assay.

Therefore, antisense inhibition of PKK mRNA may be beneficial for thetreatment and prevention of vascular permeability, which is symptomaticof HAE.

TABLE 62 OD_(600 nm) of Evans Blue dye to measure vascular permeabilityGroup No. Treatment Captopril ISIS 461756 HOE-140 Colons Intestines FeetEars 1 PBS No No No 0.26 0.16 0.11 0.02 2 PBS Yes No No 0.49 0.29 0.120.07 3 PBS Yes Yes No 0.49 0.34 0.11 0.12 4 PBS Yes Yes Yes 0.14 0.180.07 0.09 5 ISIS 141923 Yes Yes No 0.44 0.29 0.14 0.08 6 ISIS 482584 YesNo No 0.27 0.30 0.07 0.14 7 ISIS 482584 Yes Yes No 0.21 0.34 0.07 0.06

Quantification of High Molecular Weight Kininogen (HMWK)

Western blot quantification of HMWK from blood samples are presented inFIG. 1.

As shown in FIG. 1, samples from Groups 1 and 2 have low levels of HMWKas compared to Groups 6 and 7 indicating that vascular permeability isreversed in Groups 6 and 7. Also as shown in FIG. 1, samples from Groups1 and 2 have increased HMWK cleavage product as compared to Groups 6 and7. Thus, lack of HMWK is caused by PKK cleavage of HMWK into cleavageproducts (including bradykinin and HKa).

Example 12: In Vivo Effect of Antisense Inhibition of Murine PKK onBasal Permeability and Captopril-Induced Permeability in Mice

Basal permeability is the level of vascular permeability occurring inthe tissues of naïve, untreated mice. The effect of ISIS 482584 in theprevention of vascular permeability, either basal or captopril-induced,was evaluated.

Treatment

The various treatment groups for this assay are presented in Table 63.

Group 1 consisted of 8 mice and was treated with PBS administeredsubcutaneously twice a week for 4 weeks. No other treatment wasadministered to Group 1 which served as a control group to measure thebasal levels of vascular permeability.

Group 2 consisted of 8 mice and was treated with PBS administeredsubcutaneously twice a week for 4 weeks. At the end of the treatmentperiod, the mice were intraperitoneally administered 20 μg of captopril.

Group 2 served as the negative control group for captopril-inducedvascular permeability.

Group 3 consisted of 8 mice and was treated with PBS administeredsubcutaneously twice a week for 4 weeks. At the end of the treatmentperiod, the mice were intraperitoneally administered 30 μg of HOE-140.

Group 3 served as a positive control for inhibition of basal vascularpermeability.

Group 4 consisted of 8 mice and was treated with PBS administeredsubcutaneously twice a week for 4 weeks. At the end of the treatmentperiod, the mice were intraperitoneally administered 20 μg of captopril.The mice were also intraperitoneally administered 30 μg of HOE-140.Group 4 served as a positive control for inhibition of captopril-inducedvascular permeability.

Group 5 consisted of 8 mice and was treated with 40 mg/kg of ISIS 482584administered subcutaneously twice a week for 4 weeks. Group 5 served asan experimental treatment group for examining the effect of ISIS 482584on basal vascular permeability.

Group 6 consisted of 8 mice and was treated with 40 mg/kg of ISIS 482584administered subcutaneously twice a week for 4 weeks. At the end of thetreatment period, the mice were intraperitoneally administered 20 μg ofcaptopril. Group 6 served as an experimental treatment group forexamining the effect of ISIS 482584 on captopril-induced vascularpermeability.

All the groups were then injected with 30 mg/kg of Evans Blue solution.The mice were sacrificed 30 min after the Evans Blue solutionadministration and colons, feet, ears, and intestines were harvested.

TABLE 63 Treatment groups Group No. Treatment Captopril HOE-140 1. (N =8) PBS No No 2. (N = 8) PBS Yes No 3. (N = 8) PBS No Yes 4. (N = 8) PBSYes Yes 5. (N = 8) ISIS 482584 No No 6. (N = 8) ISIS 482584 Yes No

Quantification of Vascular Permeability

The harvested tissues from the feet, colon, intestine, and ears wereplaced separately in formamide solution overnight to leach out the EvansBlue dye. The formamide solution containing feet and ear tissue washeated to 55° C. and left overnight. The color intensity of thedye-infused formamide solution was then measured at OD_(600 nm), and ispresented in Table 64. Mice displaying any manifestation of angioedematake up more dye and, therefore, demonstrate high OD values.

As presented in Table 64, mice treated with ISIS 482584 demonstratedreduced basal vascular permeability compared to the PBS control (Group 5vs. Group 1). The reduction in basal vascular permeability by treatmentwith ISIS 482584 was comparable to that caused by treatment with HOE-140(Group 3, which served as the positive control). Mice treated with ISIS482584 also demonstrated reduced captopril-induced vascular permeabilityin most tissues compared to the PBS control (Group 6 vs. Group 2). Thereduction in captopril-induced vascular permeability by treatment withISIS 482584 was comparable to that caused by treatment with HOE-140(Group 4, which served as the positive control).

TABLE 64 OD_(600 nm) of Evans Blue dye to measure vascular permeabilityGroup No. Treatment Captopril HOE-140 Colon Feet Intestine Ears 1 PBS NoNo 0.27 0.08 0.23 0.06 2 PBS Yes No 0.61 0.08 0.24 0.01 3 PBS No Yes0.18 0.06 0.21 0.03 4 PBS Yes Yes 0.29 0.03 0.14 0.00 5 ISIS 482584 NoNo 0.19 0.07 0.22 0.04 6 ISIS 482584 Yes No 0.37 0.05 0.22 0.00

Example 13: Dose-Dependent Effect of Antisense Inhibition of Murine PKKon Captopril-Induced Vascular Permeability

The effect of varying doses on ISIS 482584 on captopril-induced vascularpermeability was evaluated.

Treatment

The various treatment groups for this assay are presented in Table 65.

Group 1 consisted of 4 mice and was treated with PBS administeredsubcutaneously twice a week for 3 weeks. No other treatment wasadministered to Group 1 which served as a control group to measure thebasal levels of vascular permeability.

Group 2 consisted of 8 mice and was treated with PBS administeredsubcutaneously twice a week for 3 weeks. At the end of the treatmentperiod, the mice were intraperitoneally administered 20 μg of captopril.

Group 2 served as the control group for captopril-induced vascularpermeability.

Group 3 consisted of 4 mice and was treated with PBS administeredsubcutaneously twice a week for 3 weeks. At the end of the treatmentperiod, the mice were intraperitoneally administered 20 μg of captopril.The mice were also intraperitoneally administered 30 μg of Icatibant(HOE-140). Group 4 served as a positive control for inhibition ofcaptopril-induced vascular permeability.

Groups 4, 5, 6, 7, 8, and 9 consisted of 8 mice each and were treatedwith 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 40 mg/kg, or 80 mg/kg(corresponding to 5 mg/kg, 10 mg/kg, 20 mg/kg, 40 mg/kg, 80 mg/kg, or160 mg/kg per week), respectively of ISIS 482584 administeredsubcutaneously twice a week for 3 weeks. At the end of the treatmentperiod, the mice of all the groups were intraperitoneally administered20 μg of captopril. Groups 4-9 served as the experimental treatmentgroups for examining the effect of varying doses of ISIS 482584 oncaptopril-induced vascular permeability.

All the groups were then injected with 30 mg/kg of Evans Blue solutionin the tail vein. The mice were sacrificed 30 min after the Evans Bluesolution administration and colons, feet, ears, and intestines wereharvested. Blood samples were taken through cardiac puncture.

TABLE 65 Treatment groups Dose Group No. Treatment (mg/kg/wk) CaptoprilHOE-140 1. (N = 4) PBS — No No 2. (N = 8) PBS — Yes No 3. (N = 4) PBS —Yes Yes 4. (N = 8) ISIS 482584 160 Yes No 5. (N = 8) ISIS 482584 80 YesNo 6. (N = 8) ISIS 482584 40 Yes No 7. (N = 8) ISIS 482584 20 Yes No 8.(N = 8) ISIS 482584 10 Yes No 9. (N = 8) ISIS 482584 5 Yes No

Quantification of Vascular Permeability

The harvested tissues were placed in formamide solution overnight toleach out the Evans Blue dye. The formamide solution containing feet andear tissue was heated to 55° C. and left overnight. The color intensityof the dye-infused formamide solution was then measured at OD_(600 nm),and is presented in Table 66. Mice displaying any manifestation ofangioedema take up more dye and, therefore, demonstrate high OD values.

As presented in Table 66, mice treated with higher doses of ISIS 482584(Groups 4, 5, and 6) had reduced levels of captopril-induced vascularpermeability compared to the corresponding PBS control group (Group 2).The reduction in vascular permeability in mice of these treatment groups(Groups 4 and 5) was comparable to the levels of basal vascularpermeability (as shown in Group 1) as well as in mice treated withHOE-140 (Group 3).

TABLE 66 OD_(600 nm) of Evans Blue dye to measure vascular permeabilityDose Group No. Treatment (mg/kg) Captopril HOE-140 Colon Feet IntestineEars 1 PBS — No No 0.16 0.07 0.13 0.01 2 PBS — Yes No 0.39 0.12 0.180.07 3 PBS — Yes Yes 0.15 0.03 0.10 0.04 4 ISIS 482584 160 Yes No 0.260.10 0.15 0.05 5 ISIS 482584 80 Yes No 0.21 0.04 0.17 0.03 6 ISIS 48258440 Yes No 0.36 0.10 0.20 0.05 7 ISIS 482584 20 Yes No 0.40 0.11 0.200.07 8 ISIS 482584 10 Yes No 0.41 0.10 0.19 0.05 9 ISIS 482584 5 Yes No0.41 0.10 0.17 0.05

Quantification of Vascular Leakage

The blood drawn through cardiac puncture was immediately mixed with 3times the volume of ice-cold ethanol. The solution was centrifuged at15,000 g for 20 minutes at 4° C. to remove cell debris and precipitatedplasma proteins. The ethanol extracts were further purified byultra-filtration through a 10 kDa MWCO filter. The color intensity ofthe ethanol extracted plasma solution was then measured at OD_(620 nm).The results are presented in Table 67 as percentage increase or decreaseof the OD values of the Group 1 PBS control. It was expected thattissues from mice displaying manifestation of angioedema would leak moredye from the plasma and, therefore, demonstrate low OD values, whereastreatment groups may display higher OD values due to reduced vascularleakage. Mice treated with 160 mg/kg/week and 80 mg/kg/week of ISIS482584 (Groups 4 and 5) demonstrated less vascular leakage compared tothe PBS negative control treated with captopril (Group 2). The resultsfrom Groups 4 and 5 were comparable to the positive control treated withHOE-140 (Group 3).

TABLE 67 Percentage of OD_(620 nm) of Evans Blue dye compared to the PBSbasal control to measure vascular leakage Dose Group No. Treatment(mg/kg) Captopril HOE-140 Plasma 2 PBS — Yes No −43 3 PBS — Yes Yes 5 4ISIS 482584 160 Yes No 91 5 ISIS 482584 80 Yes No 40 6 ISIS 482584 40Yes No −31 7 ISIS 482584 20 Yes No −26 8 ISIS 482584 10 Yes No −20 9ISIS 482584 5 Yes No −23

Example 14: Dose-Dependent Effect of Antisense Inhibition of Murine PKKon Basal Permeability in Mice

The effect of varying doses on ISIS 482584 on basal vascularpermeability was evaluated.

Treatment

The various treatment groups for this assay are presented in Table 68.

Group 1 consisted of 8 mice and was treated with PBS administeredsubcutaneously twice a week for 3 weeks. No other treatment wasadministered to Group 1 which served as a control group to measure thebasal levels of vascular permeability.

Group 2 consisted of 4 mice and was treated with PBS administeredsubcutaneously twice a week for 3 weeks. At the end of the treatmentperiod, the mice were intraperitoneally administered 30 μg of HOE-140.Group 2 served as a positive control for inhibition of basal vascularpermeability.

Groups 3, 4, 5, 6, 7, and 8 consisted of 8 mice each and were treatedwith 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 40 mg/kg, or 80 mg/kg(corresponding to 5 mg/kg, 10 mg/kg, 20 mg/kg, 40 mg/kg, 80 mg/kg, or160 mg/kg per week), respectively of ISIS 482584 administeredsubcutaneously twice a week for 3 weeks. Groups 4-9 served as theexperimental treatment groups for examining the effect of varying dosesof ISIS 482584 on basal vascular permeability.

All the groups were then injected with 30 mg/kg of Evans Blue solutionin the tail vein. The mice were sacrificed 30 min after the Evans Bluesolution administration and colons, feet, and ears were harvested andexamined for permeability defects. Blood samples were taken throughcardiac puncture.

TABLE 68 Treatment groups Dose Group No. Treatment (mg/kg/week)HOE-140 1. (N = 8) PBS — No 2. (N = 4) PBS — Yes 3. (N = 8) ISIS 482584160 No 4. (N = 8) ISIS 482584 80 No 5. (N = 8) ISIS 482584 40 No 6. (N =8) ISIS 482584 20 No 7. (N = 8) ISIS 482584 10 No 8. (N = 8) ISIS 4825845 No

Quantification of Vascular Permeability

The harvested tissues from the feet, colon, and ears were placed informamide solution overnight to leach out the Evans Blue dye. Theformamide solution containing feet and ear tissue was heated to 55° C.and left overnight. The color intensity of the dye-infused formamidesolution was then measured at OD_(600 nm), and is presented in Table 69.Higher OD values are associated with higher levels of permeability.

As presented in Table 10, most of the tissues of mice treated with ISIS482584 at all doses (Groups 3-8) demonstrated reduced basal vascularpermeability compared to the PBS control (Group 1). The reduction inbasal vascular permeability of the ISIS oligonucleotide-treated groupswas comparable to the same demonstrated in the positive control grouptreated with HOE-140 (Group 2).

TABLE 69 OD_(600 nm) of Evans Blue dye to measure vascular permeabilityDose HOE- Group No. Treatment (mg/kg/week) 140 Colon Feet Ears 1 PBS —No 0.27 0.17 0.013 2 PBS — Yes 0.24 0.09 0.047 3 ISIS 482584 160 No 0.250.11 0.019 4 ISIS 482584 80 No 0.24 0.09 0.014 5 ISIS 482584 40 No 0.270.11 0.011 6 ISIS 482584 20 No 0.26 0.11 0.009 7 ISIS 482584 10 No 0.310.10 0.015 8 ISIS 482584 5 No 0.32 0.11 0.009

Quantification of Vascular Leakage

The blood drawn through cardiac puncture was immediately mixed with 3times the volume of ice-cold ethanol. The solution was centrifuged at15,000 g for 20 minutes at 4° C. to remove cell debris and precipitatedplasma proteins. The ethanol extracts were further purified byultra-filtration through a 10 kDa MWCO filter. The color intensity ofthe ethanol extracted plasma solution was then measured at OD_(620 nm).The results are presented in Table 70 as percentage increase or decreaseof the OD values of the Group 1 PBS control. It was expected thattreatment groups may display higher OD values due to reduced vascularleakage. All the mice in the ISIS oligonucleotide-treated groupsdemonstrated significantly reduced vascular leakage compared to the PBSnegative control.

TABLE 70 Percentage of OD_(620 nm) of Evans Blue dye compared to the PBSbasal control to measure vascular leakage Dose Group No. Treatment(mg/kg/week) HOE-140 Plasma 2. (N = 8) ISIS 482584 160 No 95 3. (N = 8)ISIS 482584 80 No 93 4. (N = 8) ISIS 482584 40 No 83 5. (N = 8) ISIS482584 20 No 56 6. (N = 8) ISIS 482584 10 No 36

Quantification of High Molecular Weight Kininogen (HMWK)

Western blot quantification of HMWK from blood samples are presented inFIG. 2 and Tables 71 and 72.

As shown in Table 71, Groups treated with 482584 have higher levels ofHMWK as compared to PBS control, increasing in a dose-dependent manner.Treatment with PKK antisense oligonucleotide results in stabilization ofHMWK. Thus, vascular permeability is reduced in ISIS 482584-treatedgroups in a dose-dependent manner. As shown in Table 72, Groups treatedwith ISIS 482584 have lower HMWK cleavage product as compared to PBScontrol, decreasing in a dose-dependent manner. Thus, reduced HMWK iscaused by PKK cleavage of HMWK into cleavage products (includingbradykinin and HKa). Data are presented in Intensity Units as measuredby densitometer.

TABLE 71 Quantification of HMWK by densitometer Dose Group No Treatment(mg/kg/week) Intensity Units 1 PBS — 89 3 ISIS 482584 160 21358 4 ISIS482584 80 7279 5 ISIS 482584 40 873 6 ISIS 482584 20 608 7 ISIS 48258410 507

TABLE 72 Quantification of HMWK cleavage product by densitometer DoseGroup No Treatment (mg/kg/week) Intensity Units 1 PBS — 401738 3 ISIS482584 160 19936 4 ISIS 482584 80 204482 5 ISIS 482584 40 388135 6 ISIS482584 20 403360 7 ISIS 482584 10 414774

Example 15: Combination Therapy of Antisense Oligonucleotides TargetingPKK and Factor 12 on Captopril-Induced Vascular Permeability in Mice

Mice were treated varying doses of ISIS 410944, a 5-10-5 MOE gapmertargeting Factor 12 (GCATGGGACAGAGATGGTGC; SEQ ID NO: 2247), and ISIS482584 in a captopril-induced vascular permeability model.

Treatment

The various treatment groups for this assay are presented in Table 73.

Group 1 consisted of 4 mice and was treated with PBS administeredsubcutaneously twice a week for 3 weeks. No other treatment wasadministered to Group 1 which served as a control group to measure thebasal levels of vascular permeability.

Group 2 consisted of 8 mice and was treated with PBS administeredsubcutaneously twice a week for 3 weeks. At the end of the treatmentperiod, the mice were intraperitoneally administered 20 μg of captopril.

Group 2 served as the control group for captopril-induced vascularpermeability.

Group 3 consisted of 4 mice and was treated with PBS administeredsubcutaneously twice a week for 3 weeks. At the end of the treatmentperiod, the mice were intraperitoneally administered 20 μg of captopril.The mice were also intraperitoneally administered 30 μg of HOE-140.Group 3 served as a positive control for inhibition of captopril-inducedvascular permeability.

Groups 4, 5, 6, 7, and 8 consisted of 8 mice each and were treated with2.5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, or 40 mg/kg (corresponding to 5mg/kg, 10 mg/kg, 20 mg/kg, 40 mg/kg, or 80 mg/kg per week), respectivelyof ISIS 482584 and ISIS 410944 each administered subcutaneously twice aweek for 3 weeks. At the end of the treatment period, the mice of allthe groups were intraperitoneally administered 20 μg of captopril.Groups 4-8 served as the experimental treatment groups for examining theeffect of ISIS 410944 and ISIS 482584 on captopril-induced vascularpermeability.

All the groups were then injected with 30 mg/kg of Evans Blue solutionin the tail vein. The mice were sacrificed 30 min after the Evans Bluesolution administration and colons, feet, ears, and intestines wereharvested.

TABLE 73 Treatment groups Dose (mg/kg/wk) Group No. Treatment of eachASO Captopril HOE-140 1. (N = 4) PBS — No No 2. (N = 8) PBS — Yes No 3.(N = 4) PBS — Yes Yes 4. (N = 8) ISIS 482584 + 80 Yes No ISIS 410944 5.(N = 8) ISIS 482584 + 40 Yes No ISIS 410944 6. (N = 8) ISIS 482584 + 20Yes No ISIS 410944 7. (N = 8) ISIS 482584 + 10 Yes No ISIS 410944 8. (N= 8) ISIS 482584 + 5 Yes No ISIS 410944

Quantification of Vascular Permeability

The harvested tissues from the feet, colon, and ears were placed informamide solution overnight to leach out the Evans Blue dye. Theformamide solution containing feet and ear tissue was heated to 55° C.and left overnight. The color intensity of the dye-infused formamidesolution was then measured at OD_(600 nm), and is presented in Table 74.Higher OD values are associated with higher levels of permeability.

As presented in Table 74, most of the tissues of mice treated with acombination of ISIS 482584 and ISIS 410944 at all doses (Groups 3-8)demonstrated reduced vascular permeability compared to the PBS control(Group 1). The reduction in vascular permeability of the ISISoligonucleotide-treated groups was comparable to the same demonstratedin the basal PBS control (Group 1), as well as the positive controlgroup treated with HOE140 (Group 2). Combination of PKK and Factor 12antisense oligonucleotides results in synergistic decrease inpermeability. As expected, a corresponding synergistic decrease invascular leakage was also observed.

TABLE 74 OD_(600 nm) of Evans Blue dye to measure vascular permeabilityDose (mg/kg/wk) Group No. Treatment of each ASO Captopril HOE-140 ColonFeet Intestines Ears 1 PBS — No No 0.24 0.11 0.13 0.01 2 PBS — Yes No0.38 0.15 0.11 0.05 3 PBS — Yes Yes 0.23 0.06 0.15 0.04 4 ISIS 482584 +80 Yes No 0.19 0.07 0.11 0.04 ISIS 410944 5 ISIS 482584 + 40 Yes No 0.190.07 0.12 0.03 ISIS 410944 6 ISIS 482584 + 20 Yes No 0.22 0.08 0.12 0.04ISIS 410944 7 ISIS 482584 + 10 Yes No 0.38 0.13 0.13 0.05 ISIS 410944 8ISIS 482584 + 5 Yes No 0.53 0.12 0.13 0.03 ISIS 410944

Example 16: Combination Therapy of Antisense Oligonucleotides TargetingPKK and Factor 12 on Basal Vascular Permeability in Mice

Mice were treated with varying doses of ISIS 410944, an antisenseoligonucleotide targeting Factor 12, and ISIS 482584 in a basal vascularpermeability model.

Treatment

The various treatment groups for this assay are presented in Table 75.

Group 1 consisted of 8 mice and was treated with PBS administeredsubcutaneously twice a week for 3 weeks. No other treatment wasadministered to Group 1 which served as a control group to measure thebasal levels of vascular permeability.

Group 2 consisted of 4 mice and was treated with PBS administeredsubcutaneously twice a week for 3 weeks. At the end of the treatmentperiod, the mice were intraperitoneally administered 30 μg of HOE-140.

Group 2 served as a positive control for inhibition of basal vascularpermeability.

Groups 3, 4, 5, 6, and 7 consisted of 8 mice each and were treated with2.5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, or 40 mg/kg (corresponding to 5mg/kg, 10 mg/kg, 20 mg/kg, 40 mg/kg, or 80 mg/kg per week), respectivelyof ISIS 482584 and ISIS 410944 each administered subcutaneously twice aweek for 3 weeks. Groups 3-7 served as the experimental treatment groupsfor examining the effect of ISIS 410944 and ISIS 482584 on basalvascular permeability.

All the groups were then injected with 30 mg/kg of Evans Blue solutionin the tail vein. The mice were sacrificed 30 min after the Evans Bluesolution administration and colons, feet, ears, and intestines wereharvested.

TABLE 75 Treatment groups Dose Group No. Treatment (mg/kg/wk) HOE-140 1.(N = 8) PBS — No 2. (N = 4) PBS — Yes 3. (N = 8) ISIS 482584 + 80 NoISIS 410944 4. (N = 8) ISIS 482584 + 40 No ISIS 410944 5. (N = 8) ISIS482584 + 20 No ISIS 410944 6. (N = 8) ISIS 482584 + 10 No ISIS 410944 7.(N = 8) ISIS 482584 + 5 No ISIS 410944

Quantification of Vascular Permeability

The harvested tissues from the feet, colon, intestines, and ears wereplaced in formamide solution overnight to leach out the Evans Blue dye.The formamide solution containing feet and ear tissue was heated to 55°C. and left overnight. The color intensity of the dye-infused formamidesolution was then measured at OD_(600 nm), and is presented in Table 76.Higher OD values are associated with higher levels of permeability.

As presented in Table 76, most of the tissues of mice treated with acombination of ISIS 482584 and ISIS 410944 at all doses (Groups 2-7)demonstrated reduced vascular permeability compared to the PBS control(Group 1). The reduction in vascular permeability of the ISISoligonucleotide-treated groups was comparable to the same demonstratedin positive control group treated with HOE140 (Group 2). Combination ofPKK and Factor 12 antisense oligonucleotides results in synergisticdecrease in permeability. As expected, a corresponding synergisticdecrease in vascular leakage was also observed.

TABLE 76 OD_(600 nm) of Evans Blue dye to measure vascular permeabilityDose Group No. Treatment (mg/kg/wk) HOE-140 Colon Feet Intestines Ears 1PBS — No 0.19 0.08 0.10 0.004 2 PBS — Yes 0.14 0.04 0.08 0.008 3 ISIS482584 + 80 No 0.14 0.04 0.09 0.01 ISIS 410944 4 ISIS 482584 + 40 No0.15 0.05 0.10 0.006 ISIS 410944 5 ISIS 482584 + 20 No 0.15 0.04 0.100.007 ISIS 410944 6 ISIS 482584 + 10 No 0.15 0.06 0.10 0.004 ISIS 4109447 ISIS 482584 + 5 No 0.14 0.05 0.13 0.002 ISIS 410944

Example 17: Inhibition of Factor 12 Protein Activation by ISIS 482584

The effect of antisense inhibition of PKK mRNA on Factor 12 proteinactivation was evaluated.

Treatment

The various treatment groups for this assay are presented in Table 77.

Group 1 consisted of 8 mice and was treated with PBS administeredsubcutaneously twice a week for 3 weeks. No other treatment wasadministered to Group 1 which served as a control group to measureFactor 12 activation.

Groups 2, 3, 4, 5, and 6 consisted of 8 mice each and were treated with2.5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, or 40 mg/kg (corresponding to 5mg/kg, 10 mg/kg, 20 mg/kg, 40 mg/kg, or 80 mg/kg per week), respectivelyof ISIS 482584 administered subcutaneously twice a week for 3 weeks.Groups 2-6 served as the treatment groups for measuring the effect ofISIS 482584 on Factor 12 activation.

At the end of the treatment period, plasma was harvested from the micefor the Spectrozyme Factor 12a based amidolytic assay for Factor 12 inplasma.

TABLE 77 Treatment groups Dose Group No. Treatment (mg/kg/wk) 1. (N = 8)PBS — 2. (N = 8) ISIS 482584 80 3. (N = 8) ISIS 482584 40 4. (N = 8)ISIS 482584 20 5. (N = 8) ISIS 482584 10 6. (N = 8) ISIS 482584 5

Assay for Factor 12 Activation in Plasma

Plasma (5 μL) was added to 85 μL of PBS with 1 ug/ml dextran sulfate(500 kDa) in a 96 well polypropelene microplate and the solution wasincubated for 5 minutes at room temperature. Spectrozyme FXIIa (10 μL ofa 2 mM solution) and 0.2 mM KALLISTOP™ solution was added and theabsorbance kinetic was measured at 405 nm. Factor 12 activation wasmeasured in the linear phase of absorbance accumulation. The results arepresented in Table 78 as a percentage of Factor 12 activation measuredin the PBS control sample. As observed in Table 78, inhibition of PKK byISIS 482584 results in decreased activation of Factor 12 by itssubstrate, implying the that PKK is required for proper factor 12activation.

TABLE 78 Percentage Factor 12 activation compared to the PBS controlDose % F12 (mg/kg/wk) activation 80 14 40 24 20 47 10 63 5 82

Example 18: In Vivo Effect of Antisense Inhibition of Murine PKK onC1-INH Antisense Oligonucleotide-Induced Vascular Permeability

Vascular permeability induced by ISIS 461756, an antisenseoligonucleotide which targets murine C1 inhibitor mRNA, increasesvascular permeability in mice and replicates the pathology of hereditaryangioedema. The effect of ISIS 482584 on this model was evaluated.

Treatment

One group of 8 mice was treated with 40 mg/kg ISIS 482584 administeredsubcutaneously twice a week for 3 weeks (weekly dose of 80 mg/kg). Asecond group of 8 mice was treated with 40 mg/kg of the controloligonucleotide, ISIS 141923, administered subcutaneously twice a weekfor 3 weeks (weekly dose of 80 mg/kg). A third group of 8 mice wastreated with PBS administered subcutaneously twice a week for 3 weeks.On day 14, all the groups were treated with 12.5 mg/kg ISIS 461756administered subcutaneously twice a week for 3 weeks (weekly dose of 25mg/kg). A control group of mice was treated with PBS administeredsubcutaneously twice a week for 3 weeks but was not administered ISIS461756.

At the end of the treatment period, all the groups were injected with 30mg/kg of Evans Blue solution into the tail vein. The mice weresacrificed 30 min after the Evans Blue solution administration andcolons, feet, ears, and intestines were harvested. The liver was alsoharvested for RNA analysis.

RNA Analysis

RNA was isolated from the liver for RT-PCR analysis of C1-INH and PKKmRNAs. The primer probe set for C1-INH is RTS3218 (forward sequenceGAGTCCCCCAGAGCCTACAGT, designated herein as SEQ ID NO: 2234; reversesequence TGTCATTTGTTATTGTGATGGCTACA, designated herein as SEQ ID NO:2235; probe sequence CTGCCCTCTACCTGGCCAACAACCA, designated herein as SEQID NO: 2236). The primer probe set for PKK is RTS3287 (forward sequenceACAAGTGCATTTTACAGACCAGAGTAC, designated herein as SEQ ID NO: 2237;reverse sequence GGTTGTCCGCTGACTTTATGCT, designated herein as SEQ ID NO:2238; probe sequence AAGCACAGTGCAAGCGGAACACCC, designated herein as SEQID NO: 2239). The results are presented in Table 79 as percentinhibition compared to the PBS control not treated with ISIS 461756. Thedata indicates that ISIS 461756 significantly reduced C1-INH mRNAexpression and that treatment with ISIS 482584 significantly reduced PKKexpression.

TABLE 79 Percent inhibition of mRNA expression in mice treated with ISIS461756 compared to the untreated PBS control C1-INH PKK Treatment mRNAmRNA PBS 76 0 ISIS 141923 79 0 ISIS 482584 77 78

Quantification of Vascular Permeability

The harvested tissues from the feet, colon, and intestines were placedin formamide solution overnight to leach out the Evans Blue dye. Theformamide solution containing feet tissue was heated to 55° C. and leftovernight. The color intensity of the dye-infused formamide solution wasthen measured at OD_(600 nm) The data is presented in Table 80 aspercent increase or reduction compared to the PBS control not treatedwith ISIS 461756. The data indicates that treatment with ISIS 482584prevented vascular permeability induced by ISIS 461756.

TABLE 80 Percent change in vascular permeability in mice treated withISIS 461756 compared to the untreated PBS control Treatment Colon FeetIntestines PBS 13 70 27 ISIS 141923 2 80 14 ISIS 482584 −23 2 −25

Example 19: In Vivo Effect of Antisense Inhibition of Murine PKK in theFeCl₃-Induced Inferior Vena Cava Thrombosis Model

ISIS 482584, which demonstrated significant in vitro and in vivoinhibition of PKK, was evaluated in the FeCl₃-induced inferior vena cavathrombosis mouse model.

Treatment

Three groups of 8 male BALB/c mice were treated with 10 mg/kg, 20 mg/kg,or 40 mg/kg of ISIS 482584, administered subcutaneously twice a week for3 weeks (weekly doses of 20 mg/kg, 40 mg/kg, or 80 mg/kg). Two controlgroups of 12 BALB/c mice each were treated with PBS, administeredsubcutaneously twice a week for 3 weeks. Two days after the last dose ofantisense oligonucleotide or PBS, mice from all groups were anesthetizedwith 150 mg/kg ketamine mixed with 10 mg/kg xylazine, administered byintraperitoneal injection. Thrombus formation was induced with FeCl₃ inall groups of anesthetized mice except the first control group.

In mice undergoing FeCl₃ treatment, thrombus formation was induced byapplying a piece of filter paper (2×4 mm) pre-saturated with 10% FeCl₃solution directly on the vena cava. After 3 minutes of exposure, thefilter paper was removed. Thirty minutes after the filter paperapplication, a fixed length of the vein containing the thrombus wasdissected out for platelet analysis. Liver was collected for RNAanalysis.

Quantification of Platelet Composition

Real-time PCR quantification of platelet factor-4 (PF-4) was used toquantify platelets in the vena cava as a measure of thrombus formation.PF-4 mRNA levels were measured using the murine primer probe setmPF4_LTS_00086 (forward sequence AGACCCATTTCCTCAAGGTAGAACT, designatedherein as SEQ ID NO: 2240; reverse sequence CGCAGCGACGCTCATG, designatedherein as SEQ ID NO: 2241; probe sequence TCTTTGGGTCCAGTGGCACCCTCTT,designated herein as SEQ ID NO: 2242). Results are presented as apercentage of PF-4 in ISIS oligonucleotide-treated mice, as compared tothe two PBS-treated control groups. As shown in Table 81, treatment withISIS 482584 resulted in a significant reduction of PF-4 in comparison tothe PBS control. Therefore, reduction of PKK by the compound providedherein is useful for inhibiting thrombus formation.

TABLE 81 Analysis of thrombus formation by real-time PCR quantificationof PF-4 in the FeCl₃ induced venous thrombosis model Dose in mg/kg/wkPF-4 PBS − FeCl₃ — 0 PBS + FeCl₃ — 100 ISIS 482584 20 62 40 34 80 25

Example 20: In Vivo Effect of Antisense Inhibition of Murine PKK in aTail Bleeding Assay

Tail-bleeding was measured to observe whether treatment with ISIS 482584causes excess bleeding or hemorrhage in mice.

Treatment

Groups of 10 male BALB/c mice were treated with 10 mg/kg, 20 mg/kg, or40 mg/kg of ISIS 482584, administered subcutaneously twice a week for 3weeks (weekly doses of 20 mg/kg, 40 mg/kg, or 80 mg/kg). A control groupof 8 BALB/c mice was treated with PBS, administered subcutaneously twicea week for 3 weeks.

Tail-Bleeding Assay

Two days after the final treatment of ISIS oligonucleotides or PBS, micewere placed in a tail bleeding chamber. Mice were anesthetized in thechamber with isoflurane. Then, a small piece of tail (approximately 4 mmfrom the tip) was cut with sterile scissors. The cut tail wasimmediately placed in a 15 mL Falcon tube filled with approximately 10mL of 0.9% NaCl buffer solution warmed to 37° C. The blood was collectedover the course of 40 minutes. The saline filled tubes were weighed bothbefore and after bleeding. The results are provided in Table 82.

Treatment with ISIS 482584 did not significantly affect bleeding. Thesedata suggest that the hemorrhagic potential of the compounds providedherein is low. These data taken with the results provided in Example 19suggest inhibition of PKK with the compounds described herein are usefulfor providing antithrombotic activity without associated bleeding risk.

TABLE 82 Tail bleeding assay after treatment with ISIS 482584 DoseBleeding (mg/kg/wk) (mL) PBS — 0.03 ISIS 482584 20 0.03 40 0.14 80 0.07

Example 21: In Vivo Effect of Antisense Inhibition of Murine PKK in theFeCl₃ Induced Mesenteric Thrombosis Model

ISIS 482584 was evaluated in the FeCl₃ induced mesenteric thrombosismouse model.

Treatment

A group of 6-8 Swiss-Webster mice was treated with 40 mg/kg of ISIS482584, administered subcutaneously twice a week for 3 weeks (weeklydose of 80 mg/kg). A control group of 6 Swiss-Webster mice was treatedwith PBS, administered subcutaneously twice a week for 3 weeks. Two daysafter the last dose of antisense oligonucleotide or PBS, mice from allgroups were anesthetized with 75 mg/kg ketamine mixed with 25 mg/kgxylazine, administered by subcutaneous injection.

Rhodamine 6G dye at a dosage of 5 mg/kg was injected subcutaneously tostain platelets. Alexa-647-labeled anti-fibrinogen antibody at a dosageof 1 mg/kg was injected via tail vein injection to stain fibrin. Theabdomen was opened by a middle incision. The visceral mesentery wasspread on a glass coverslip and the mesenteric arterioles (70-120 μm)were located by observation under a microscope. Thrombus formation wasinduced by applying of cotton threads (2×0.3 mm) pre-saturated with 6%FeCl₃ solution directly on the target vessel. After three minutes ofexposure, the thread was removed and the color intensities of both thedyes were recorded by fluorescent microscopy (Olympus FluoView 1000confocal laser scanning microscope) with appropriate filters for 70 min.

The results for platelet aggregation in the control and treatment groupsare presented in Table 83, expressed in arbitrary units (a.u.). Plateletaggregation was reduced in mice treated with ISIS 482584 at a dose of 80mg/kg/week as compared to mice treated with PBS. The results for fibrinformation in the control and treatment groups are presented in Table 84,also expressed in arbitrary units (a.u.). Fibrin formation was reducedin mice treated with ISIS 482584 at a dose of 80 mg/kg/week as comparedto mice treated with PBS. Therefore, these results suggest that ISIS482584 inhibits thrombus formation.

TABLE 83 Analysis of platelet aggregation by real-time measurement offluorescent intensity (a.u.) in a FeCl₃ induced mesenteric thrombusmodel Time 80 (sec) PBS mg/kg/wk 752 54 74 1018 315 11 1284 485 7 1550654 0 1815 1079 0 2081 1164 0 2347 1452 0 2613 1440 38 2879 1689 1483144 1716 129 3410 1845 169 3676 1865 131 3944 2055 87

TABLE 84 Analysis of fibrin formation by real-time measurement offluorescent intensity (a.u.) in a FeCl₃ induced mesenteric thrombusmodel Time 80 (sec) PBS mg/kg/wk 752 9 54 1018 86 7 1284 203 1 1550 31910 1815 521 16 2081 598 15 2347 831 61 2613 959 88 2879 1157 141 31441236 150 3410 1374 173 3676 1629 160 3944 1822 128

Example 22: In Vivo Effect of Antisense Inhibition of Murine PKK in theStenosis-Induced Inferior Vena Cava Thrombosis Model

ISIS 482584 was evaluated in the stenosis-induced inferior vena cava(IVC) thrombosis model. Reduced blood flow and endothelial damage arehallmarks of this model, also known as the St. Tomas model.

Treatment

Four groups of 6-8 BALB/c mice were treated with 5 mg/kg, 10 mg/kg, 20mg/kg, or 40 mg/kg of ISIS 482584, administered subcutaneously twice aweek for 3 weeks (weekly doses of 10 mg/kg, 20 mg/kg, 40 mg/kg, or 80mg/kg). A control group of 8 BALB/c mice was treated with PBS,administered subcutaneously twice a week for 3 weeks. Two days after thelast dose of antisense oligonucleotide or PBS was administered, micefrom all groups were anesthetized with 2.5% inhalant isoflurane. The IVCof the mice was exposed via a midline abdominal incision below the leftrenal vein, and was separated from the abdominal aorta by bluntdissection. A 6-0 silk tie (Ethicon, UK) was placed behind the bloodvessel just below the left renal vein and a metal 4-0 suture (Ethicon,UK) was placed longitudinally over the IVC to tie the silk tie on top.The metal suture was then removed. Two neurovascular surgical clips(Braun Medical Inc, PA) were placed at two separate positions below theligation for 20 seconds each, after which they were removed. Theabdominal cavity contents were then replaced and the abdomen was closed.After 24 hrs, the IVC was exposed and checked for thrombi formation. Allthrombi formed were collected and fixed in 10% formalin for 24 hrs.

The thrombi were weighed and the results are presented in Table 85,expressed in milligrams. As demonstrated by the results, treatment withincreasing doses of ISIS 482584 resulted in corresponding decrease inthrombus weight. The results indicate that antisense inhibition of PKKis useful for inhibiting thrombus formation.

TABLE 85 Thrombi weights in the stenosis-induced IVC thrombosis modelDose in Weight mg/kg/wk (mg) PBS — 10 ISIS 482584 10 8 20 6 40 5 80 3

1.-21. (canceled)
 22. A compound, comprising a modified oligonucleotideconsisting of 12 to 30 linked nucleosides and having a nucleobasesequence comprising at least 8 consecutive nucleobases of any of thenucleobase sequences of SEQ ID NOs: 334, 495, 587, 598, 635, 1349, 1352,1389, 1516, 1544, 1548, 1569, 1617, 1804, 1881, and
 2019. 23-33.(canceled)
 34. The compound of claim 22, wherein the nucleobase sequenceof the modified oligonucleotide is complementary to and equal lengthportion of SEQ ID NO:
 10. 35. The compound of claim 22, wherein themodified oligonucleotide is a single-stranded modified oligonucleotide.36. The compound of claim 22, wherein at least one internucleosidelinkage is a modified internucleoside linkage.
 37. The compound of claim36, wherein the at least one modified internucleoside linkage is aphosphorothioate internucleoside linkage.
 38. The compound of claim 22,wherein the modified oligonucleotide has a nucleobase sequencecomprising at least 12 consecutive nucleobases of any of the nucleobasesequences of SEQ ID NOs: 334, 495, 587, 598, 635, 1349, 1352, 1389,1516, 1544, 1548, 1569, 1598, 1617, 1665, 1666, 1804, 1881, and 2019.39. The compound of claim 22, wherein at least one nucleoside comprisesa modified nucleobase.
 40. The compound of claim 39, wherein themodified nucleobase is a 5-methylcytosine.
 41. The compound of claim 22,wherein the modified oligonucleotide comprises at least one modifiedsugar.
 42. The compound of claim 41, wherein the modified sugar is a 2′modified sugar, a BNA, or a THP.
 43. The compound of claim 42, whereinthe modified sugar is any of a 2′-O-methoxyethyl, 2′-O-methyl, aconstrained ethyl, a LNA, or a 3′-fluoro-HNA.
 44. (canceled)
 45. Thecompound of claim 22, wherein the modified oligonucleotide comprises: agap segment consisting of 10 linked deoxynucleosides; a 5′ wing segmentconsisting of 5 linked nucleosides; and a 3′ wing segment consisting of5 linked nucleosides; wherein the gap segment is positioned between the5′ wing segment and the 3′ wing segment and wherein each nucleoside ofeach wing segment comprises a modified sugar.
 46. The compound of claim1, wherein the modified oligonucleotide is conjugated to a carbohydrate.47-54. (canceled)
 55. A composition comprising the compound of claim 22and at least one of a pharmaceutically acceptable carrier or diluent.56. A method comprising administering to an animal the composition ofclaim
 55. 57. The method of claim 56, wherein the animal is a human. 58.The method of claim 57, wherein administering the compound prevents,treats, or ameliorates a PKK associated disease, disorder or condition.59. The method of claim 58, wherein the PKK associated disease, disorderor condition comprises edema.
 60. (canceled)
 61. The method of claim 57,wherein the PKK associated disease, disorder or condition comprises atleast one of a thrombosis and an embolism.
 62. The composition of claim55, wherein the pharmaceutically acceptable carrier or diluent isphosphate-buffered saline.